EP4194560A1 - Improved production of secreted proteins in fungal cells - Google Patents

Improved production of secreted proteins in fungal cells Download PDF

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EP4194560A1
EP4194560A1 EP21213084.3A EP21213084A EP4194560A1 EP 4194560 A1 EP4194560 A1 EP 4194560A1 EP 21213084 A EP21213084 A EP 21213084A EP 4194560 A1 EP4194560 A1 EP 4194560A1
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protein
yeast
gene
cell
fungal
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French (fr)
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Lars Steinmetz
S. Andreas JOHANSSON
Thomas Desfougeres
Thierry Dulermo
Georges Pignede
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Europaisches Laboratorium fuer Molekularbiologie EMBL
Lesaffre et Cie SA
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Europaisches Laboratorium fuer Molekularbiologie EMBL
Lesaffre et Cie SA
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Priority to EP21213084.3A priority Critical patent/EP4194560A1/en
Priority to PCT/EP2022/084820 priority patent/WO2023104896A1/en
Priority to PCT/EP2022/084829 priority patent/WO2023104902A1/en
Publication of EP4194560A1 publication Critical patent/EP4194560A1/en
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    • C12R2001/885—Trichoderma

Definitions

  • the present invention relates to a yeast or filamentous fungal cell producing at least one secreted protein of interest, wherein said cell comprises at least one additional fungal gene showing increased expression and/or overexpression, showing reduced expression and/or inactivation, wherein said gene improves the production of the at least one secreted protein of interest.
  • the present invention further relates to respective methods for production and uses of the yeast or filamentous fungal cells.
  • enzymes in a heterologous host allows (i) the production of enzymes of interest from slow growing or even non-cultivable organisms, (ii) the much higher production of the enzyme of interest, (iii ) the production of proteins from pathogenic or toxin-producing organisms, and (iv) the increase of the stability or activity of an enzyme by protein engineering (Falch, 1991; Demain and Vaishnav, 2009).
  • microorganisms including filamentous fungi (Aspergillus sp., Trichoderma sp.), yeasts (for example Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica) or bacteria (for example Escherichia coli, Bacillus sp.), are used to produce recombinant proteins (Demain and Vaishnav, 2009).
  • filamentous fungi Aspergillus sp., Trichoderma sp.
  • yeasts for example Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica
  • bacteria for example Escherichia coli, Bacillus sp.
  • S . cerevisiae Compared to other organisms conventionally used to produce recombinant proteins, S . cerevisiae has the advantage of rapid growth, easy manipulation both at the genetic level and at the level of production in bioreactors, and having Generally Recognized As Safe (GRAS) status.
  • GRAS Generally Recognized As Safe
  • the production of a heterologous target protein in yeast host cells is further advantageous in that it allows the target proteins to be folded and secreted through the cellular secretory machinery.
  • Yeast is already widely used for many industrial applications (breadmaking, production of drinking alcohol and biofuels, etc. Parapouli et al., 2020) where it may be advantageous to have it produce heterologous enzymes.
  • the commercialized yeast strains of S. cerevisiae secrete enzymatic activities allowing the degradation of industrial mashes containing starch derivatives. This allows bioethanol manufacturers to limit their intake of exogenous enzymes and reduce their production costs.
  • US 2011-0129872A1 relates to a method for producing a recombinant protein, comprising culturing a yeast transformed with a recombinant gene construct comprising a yeast promoter, a gene coding a signal sequence and a gene coding a target protein; and also with one or more genes coding folding accessory protein selected from the group consisting of PDI1 (protein disulfide isomerase 1), SEC23 (secretory 23), TRX2 (thioredoxin 2) AHA1 (activator of heat shock protein 90 ATPase), and SCJ1 (S. cerevisiae DnaJ), followed by culturing the transformed yeast.
  • PDI1 protein disulfide isomerase 1
  • SEC23 secretory 23
  • TRX2 thioredoxin 2
  • AHA1 activator of heat shock protein 90 ATPase
  • SCJ1 S. cerevisiae DnaJ
  • US 2013-0011875 relates to a method and the production of higher titers of recombinant protein in a modified yeast host cell, for example Pichia pastoris, wherein the modified yeast cell lacks vacuolar sorting activity or has decreased vacuolar sorting activity relative to an unmodified yeast host cell of the same species.
  • US 2014-0335622 discloses an expression vector for secreting a protein (Z) to be recovered or a fusion protein having the protein (Z) moiety therein; a method for producing a transformant using the expression vector; the transformant; and a method for producing a protein using the transformant. It is disclosed that co-expression of a foreign secretory protein with PDI1 increases the secretory production amount.
  • US 2016-0186192 describes a method for producing a desired protein comprising: (a) providing a host cell comprising a first recombinant gene encoding a protein comprising the sequence of a first chaperone protein, a second recombinant gene encoding a protein comprising the sequence of a second chaperone protein and a third gene, such as a third recombinant gene, encoding a desired protein (such as a desired heterologous protein), wherein the first and second chaperones are different; and (b) culturing the host cell in a culture medium to obtain expression of the first, second and third genes.
  • US 2018-0022785 claims a method for producing a heterologous protein, said method comprising: culturing a Saccharomyces cerevisiae yeast host cell or a culture thereof to produce the heterologous protein, wherein said Saccharomyces cerevisiae yeast host cell comprises a modified Not4 protein, and wherein said heterologous protein is an albumin, or a variant, fragment and/or fusion thereof.
  • Eun Jung Thak et al. discloses that yeasts are prominent hosts for the production of recombinant proteins from industrial enzymes to therapeutic proteins. Particularly, the similarity of protein secretion pathways between these unicellular eukaryotic microorganisms and higher eukaryotic organisms has made them a preferential host to produce secretory recombinant proteins.
  • Zihe Liu, etal. discloses that the increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production.
  • amylase secretion could be improved by 35%.
  • transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.
  • Mutated genes identified are disclosed to be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion.
  • the combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.
  • Huang M, et al. in: Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production. Proc Natl Acad Sci U S A. 2018 Nov 20;115(47):E11025-E11032. doi: 10.1073/pnas.1809921115. Epub 2018 Nov 5. PMID: 30397111; PMCID: PMC6255153 ) describe that baker's yeast Saccharomyces cerevisiae is one of the most important and widely used cell factories for recombinant protein production.
  • Cryptic unstable transcripts are a subset of non-coding RNAs (ncRNAs) that are produced from intergenic and intragenic regions. Additionally, stable uncharacterized transcripts, or SUTs, have also been detected in cells and bear many similarities to CUTs but are not degraded through the same pathways.
  • cerevisiae remains relatively limited in its ability to secrete proteins compared to organisms such as filamentous fungi or P. pastoris (Demain and Vaishnav, 2009). It is therefore an object of the present invention to provide new factors to improve recombinant protein production and secretion in yeast. Other objects and advantages will become apparent to the person of skill when studying the present description of the present invention.
  • the above object is solved by providing a yeast or filamentous fungal cell producing at least one secreted protein of interest, wherein said cell comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, VHS2, ASA1, TRP4, YPS7, CUT824, YOR318C, PRM7, ERV46, FIT2, GPM3, CUT892, SRN2, SUT643, CUT461, THR4, GMH1, SOL1, NAB6, YPR148C, ALP1, CUT097, ATG33, YOR316C-A, SOG2, MCM6, SUT230, SUT419, TIF11, TAF5, PHO91, AIM32, ENO2, UBA2, PUS5, ERG1, SUT311, KSS1, MRP10, CUT598, CUT188, YOR238W, E
  • said cell comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1, RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, and WBP1, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, NDC1, PET100, NIP7, VHT1, and SUT685, wherein said at least one fungal gene shows reduced expression and/or inactivation.
  • yeast or filamentous fungal cell wherein said genes or SUTs or CUTs are furthermore selected from the group of genes or SUTs or CUTs having a value of logFC/FDR of more than 200, more preferred of more than 300, and most preferred of more than 500, based on the values as determined herein.
  • yeast or filamentous fungal cell further comprising a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions, such as, without wanting to be bound by theory, for example, the impact of CRISPRa and CRISPRi on gene expression due to the position of the gRNA in the promoting region.
  • a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions, such as, without wanting to be bound by theory, for example, the impact of CRISPRa and CRISPRi on gene expression due to the position of the gRNA in the promoting region.
  • yeast or filamentous fungal cell according to the present invention further comprising the fungal gene PDI1, showing an increased expression and/or overexpression.
  • the yeast or filamentous fungal cell according to the present invention produces the at least one secreted protein to about 30% or more, or about 40% or more, preferably about 50% or more, more preferably to about 75% or more, when compared to a control yeast or filamentous fungal cell.
  • a method for producing a secreted protein in a yeast or filamentous fungal cell comprising the steps of i) providing a yeast or filamentous fungal cell producing at least one secreted protein of interest according to the present invention, ii) culturing said yeast or filamentous fungal cell in suitable culture medium, and iii) isolating said secreted protein from aid culture medium.
  • the method according to the present invention wherein about 30% or more, or about 40% or more, preferably about 50% or more, more preferably to about 75% or more of said at least one secreted protein is produced, when compared to the production of a control yeast or filamentous fungal cell.
  • the above object is solved by a method for producing a yeast or filamentous fungal cell producing at least one secreted protein of interest according to the present invention, comprising introducing into said cell producing at least one secreted protein of interest at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, VHS2, ASA1, TRP4, YPS7, CUT824, YOR318C, PRM7, ERV46, FIT2, GPM3, CUT892, SRN2, SUT643, CUT461, THR4, GMH1, SOL1, NAB6, YPR148C, ALP1, CUT097, ATG33, YOR316C-A, SOG2, MCM6, SUT230, SUT419, TIF11, TAF5, PHO91, AIM32, ENO2, UBA2, PUS5, ERG1, SUT311, KSS
  • the method according to the invention may include further introducing into said cell a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions. Furthermore, the method may include further introducing into said cell the fungal gene PDI1, showing an increased expression and/or overexpression.
  • the above object is solved by the use of a yeast or filamentous fungal cell according to the present invention for producing at least one secreted protein of interest.
  • UV S. cerevisiae mutants expressing an ⁇ -amylase has revealed improved strains for secretion (Huang et al., 2015; Huang et al., 2018). Coupling microfluidics with a phenotypic screening using a starch complexed with BODIPY (which becomes fluorescent when it is released), the authors had selected the mutants secreting the most enzyme into the extracellular medium.
  • the sequencing of eight hypersecretory clones revealed 330 mutations potentially involved in improving ⁇ -amylase production and secretion (Huang et al., 2015).
  • a more in-depth analysis led to the identification of - amongst others as disclosed herein - a role of the known PDI1 gene in the production and secretion of ⁇ -amylase in S. cerevisiae.
  • the purpose of the present invention was to discover new factors and genes involved in protein secretion in order to improve protein production and secretion, as exemplified in the industrial Ethanol Red ® strain of S. cerevisiae.
  • a yeast or filamentous fungal cell that produces at least one secreted protein of interest.
  • the cell comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, VHS2, ASA1, TRP4, YPS7, CUT824, YOR318C, PRM7, ERV46, FIT2, GPM3, CUT892, SRN2, SUT643, CUT461, THR4, GMH1, SOL1, NAB6, YPR148C, ALP1, CUT097, ATG33, YOR316C-A, SOG2, MCM6, SUT230, SUT419, TIF11, TAF5, PHO91, AIM32, ENO2, UBA2, PUS5, ERG1, SUT311, KSS1, MRP10, CUT598, CUT188, YOR238W, E
  • the terms “increased expression” or “overexpression” indicate that the amount of protein as produced by the cell is higher when compared to the expression in a control cell showing normal, unaltered or baseline expression.
  • the change in expression can be achieved in any suitable way, and examples include mutated promotors, cloning of the gene under the control of a heterologous "strong” promotor, either inducible or constitutive, codon optimization, and mutations that stabilize the structure of the protein, and the like.
  • the cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, YDR262W, CMC1, MRP17, YPT52, CUT312, MRPS5, RDR1, DAL7, RPL20A, YBR137W, RPL36B, YEL008C-A, RAX1, INP51, CUT729, UBP8, CUT258, YLR342W-A, SUT568, PEX7, MSD1, CUT136, TIM10, CUT361, snR51, TAL1, RIP1, MRP10, SUT078, MRP51, GLO3, EHD3, HER1, NMA111, PBP4, MFB1, IKI3, NDL1, SUT433, YOR238W, SUT750, QDR2, RDI1, SUT014, CUT437, MSC6, SUT497, YCR051W, MRPL33, RPL14A, TRM7
  • the terms “reduced expression” or “inactivation” indicate that the amount of protein as produced by the cell is lower when compared to the expression in a control cell showing normal, unaltered or baseline expression.
  • the change in expression can be achieved in any suitable way, and examples include mutated promotors, cloning of the gene under the control of a heterologous "weak” promotor, either inducible or constitutive, codon changes, and mutations that de-stabilize the structure of the protein, and the like.
  • said yeast or filamentous fungal cell as provided comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1, RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, and WBP1, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, NDC1, PET100, NIP7, VHT1, and SUT685, wherein said at least one fungal gene shows reduced expression and/or inactivation.
  • yeast or filamentous fungal cell wherein said genes or SUTs or CUTs are furthermore selected from the group of genes or SUTs or CUTs having a value of logFC/FDR of more than 200, more preferred of more than 300, and most preferred of more than 500, based on the values as determined herein.
  • yeast or filamentous fungal cell according to the present invention, further comprising a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions.
  • yeast or filamentous fungal cell according to the present invention further comprising the fungal gene PDI1, showing an increased expression and/or overexpression.
  • TLG2, YDR262W, and TRM10 are all of TLG2, YDR262W, and TRM10 , optionally further comprising PDI1.
  • Other examples are ATG33 and MRP10, NDC1 and TRM10 , or PRY2, and TOM22, again each pair optionally further comprising PDI1.
  • AVT2 Most preferred are either AVT2 , PRY2, SUT074, BNA7, TOM22 or TRM10.
  • the overexpression of AVT2, TRM10, PRY2, SUT074, BNA7, or TOM22, and the inactivation of INP51 is further preferred.
  • Further examples are TLG2, CUT901, ATG33, THR4, YDR262W, and CMC1, optionally further comprising PDI1.
  • the fungal gene(s) and/or SUTs or CUTs as used are preferably derived from S. cerevisiae, or a related yeast.
  • the fungal gene(s) and/or SUTs or CUTs and their reference numbers are according to the Saccharomyces Genome Database ( SGD ) (https://www.yeastgenome.org/), as of November 15, 2021.
  • Related genes that may be used as well encode for proteins sharing the same biological effect (increased secretion) in the yeast or filamentous fungal cell with the genes as above, and/or have an amino acid identity of about 80% or more, preferably about 90% or more, more preferably about 95% or more with the polypeptide sequence as encoded by a genes as above.
  • the yeast or filamentous fungal cell according to the present invention produces the at least one secreted protein to about 30% or more or 40% or more, preferably about 50% or more, more preferably to about 75% or more, when compared to a control yeast or filamentous fungal cell, preferably one that does not contain a gene as mentioned above leading to increased secretion of the protein of interest.
  • any protein can be chosen that can be suitably produced by the yeast or filamentous fungal cell according to the present invention, e.g. expressed, folded, glycosylated and/or secreted.
  • the gene of the protein of interest can be codon optimized, and preferably show an increased expression and/or overexpression, as explained above for the fungal gene according to the present invention.
  • preferred proteins of interest are human serum albumin (HSA), amylase, human insulin, and components of hepatitis vaccines, human papillomavirus (HPV) vaccines, interferon(s), or epidermal growth factor (hEGF), and proteins used in food production, such as cellulase, glucoamylase, xylanase, and the like.
  • CRISPRi and CRISPRa libraries allowing the overexpression or the repression of all genes as well as previously identified Stable Unannotated Transcripts (SUT's) and (Cryptic Unstable Transcripts CUT's) of this yeast (see Xu, Z. et al. Bidirectional promoters generate pervasive transcription in yeast. Nature 457, 1033-1037 (2009 )).
  • CRISPRa inactivated Cas9
  • VPRi VP64-p65-Rta
  • the industrial Ethanol Red ® (ER) yeast strain overexpressing an ⁇ -amylase ( Amy6 from A. niger ) was used as a model for the present invention (Lesaffre, Marcq-en-Bar ⁇ ul, France).
  • the ER + ⁇ -amylase strain was then transformed using the CRISPRa and CRISPRi libraries, and the cell population as obtained was screened by microfluidics on the basis of its capacity to degrade a starch substrate labelled with BODIPY FL dye which fluoresces in green when the starch is degraded by ⁇ -amylase (e.g. EnzChek ® Ultra Amylase Assay Kit: https://www.thermofisher.com/order/catalog/product/E33651#/E33651).
  • BODIPY FL dye which fluoresces in green when the starch is degraded by ⁇ -amylase
  • ⁇ -amylase activity was evaluated in the respective strains.
  • the overexpression of BNA7, SUT074, TOM22, TLG2, YDR262W, ALP1, ENO2 , NMA2, PRY2, and INP51 were identified as preferred for the exemplary ⁇ -amylase secretion in the Ethanol Red ® strain.
  • any suitable cell of a yeast or filamentous fungus can be used for the production of the protein of interest according to the present invention.
  • said yeast or filamentous fungal cell is selected from the group consisting of Aspergillus spp., Trichoderma spp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces ssp., Pichia spp., Hansenula polymorpha, Fusarium spp., Neurospora spp., and Penicillium spp., preferably Saccharomyces cerevisiae.
  • the at least one fungal gene showing increased expression and/or overexpression and/or showing reduced expression and/or inactivation is a native gene and/or is a recombinant gene, i.e. a modified gene of the yeast or filamentous fungal cell itself, or at least one gene that is recombinantly introduced and may be a heterologous gene, i.e. coming from a different strain or fungal species.
  • the recombinant gene is integrated into the genome as an expression cassette.
  • Respective expression cassettes for fungal expression are known, and basically consist of a promoter, the fungal gene, and a terminator.
  • the gene can be extrachromosomally expressed, preferably using a replicative expression vector, such as a shuttle vector. Promoters used in yeast and fungal expression systems are usually either inducible or constitutive.
  • the folding and glycosylation of the secretory proteins in the endoplasmatic reticulum (ENDR) is assisted by numerous ENDR-resident proteins.
  • the chaperones like Bip (GRP78), GRP94 or yeast Lhs1p help the secretory protein to fold by binding to exposed hydrophobic regions in the unfolded states and preventing unfavourable interactions ( Blond-Elguindi et al., 1993, Cell 75:717-728 ).
  • the chaperones are also important for the translocation of the proteins through the ENDR membrane.
  • the proteins like protein disulphide isomerase and its homologs and prolyl-peptidyl cis-trans isomerase assist in formation of disulphide bridges and formation of the right conformation of the peptide chain adjacent to proline residues, respectively.
  • a machinery including many protein components also resides in the ENDR for the addition of the N-linked core glycans to the secretory protein and for the initial trimming steps of the glycans.
  • yeast or filamentous fungal cell according to the present invention, wherein the cell furthermore comprises at least one additional recombinant secretion promoting gene, for example a fungal gene for a chaperone, for a foldase and/or for a glycosylation-promoting protein.
  • additional recombinant secretion promoting gene for example a fungal gene for a chaperone, for a foldase and/or for a glycosylation-promoting protein.
  • these proteins may be controllably expressed, inducible, constitutive, and even overexpressed.
  • yeast or filamentous fungal cell according to the present invention, wherein the increased expression and/or overexpression or reduced expression and/or inactivation of the at least one fungal gene or the at least one additional recombinant secretion promoting gene is constitutive or inducible.
  • Another important aspect of the present invention relates to a method for producing a secreted protein in a yeast or filamentous fungal cell, comprising the steps of i) providing a yeast or filamentous fungal cell producing at least one secreted protein of interest according to the present invention as above, ii) suitably culturing said yeast or filamentous fungal cell in suitable culture medium, and iii) isolating said secreted protein from said culture medium.
  • Methods for isolating proteins from cultures are known by the person of skill.
  • Culturing methods for producing proteins in yeast or filamentous fungal cells are known by the person of skill, and can be readily adjusted to the present invention. Culturing can be continuous or in batches or fed-batches. Preferred is the method according to the present invention, further comprising suitably inducing the increased expression and/or overexpression or reduced expression and/or inactivation of the at least one fungal gene. Induction can be achieved based on the promotor(s) as used, e.g. by adding inducers, or switching conditions, e.g. temperature.
  • Another important aspect of the present invention relates to a method for producing a yeast or filamentous fungal cell producing at least one secreted protein of interest, comprising introducing into said cell producing at least one secreted protein of interest at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, VHS2, ASA1, TRP4, YPS7, CUT824, YOR318C, PRM7, ERV46, FIT2, GPM3, CUT892, SRN2, SUT643, CUT461, THR4, GMH1, SOL1, NAB6, YPR148C, ALP1, CUT097, ATG33, YOR316C-A, SOG2, MCM6, SUT230, SUT419, TIF11, TAF5, PHO91, AIM32, ENO2, UBA2, PUS5, ERG1, SUT311, KSS1, MRP10, CUT598, C
  • the method according to the invention may include further introducing into said cell a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions.
  • said method further comprises introducing into said cell the fungal gene PDI1, showing an increased expression and/or overexpression.
  • said at least one fungal gene is also integrated into the genome as an expression cassette and/or extrachromosomally expressed, preferably using a replicative expression vector
  • Another important aspect of the present invention relates to the use of a yeast or filamentous fungal cell according to the present invention for producing at least one secreted protein of interest, preferably using a method according to the present invention.
  • the present invention provides the following items.
  • a yeast or filamentous fungal cell producing at least one secreted protein of interest, wherein said cell comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, VHS2, ASA1, TRP4, YPS7, CUT824, YOR318C, PRM7, ERV46, FIT2, GPM3, CUT892, SRN2, SUT643, CUT461, THR4, GMH1, SOL1, NAB6, YPR148C, ALP1, CUT097, ATG33, YOR316C-A, SOG2, MCM6, SUT230, SUT419, TIF11, TAF5, PHO91, AIM32, ENO2, UBA2, PUS5, ERG1, SUT311, KSS1, MRP10, CUT598, CUT188, YOR238W, EMW1, BNA7, SNR63, CCT3, PRY
  • Item 2 The yeast or filamentous fungal cell according to Item 1, wherein said cell comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1, RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, and WBP1, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, NDC1, PET100, NIP7, VHT1, and SUT685, wherein said at least one fungal gene shows reduced expression and/or inactivation.
  • said at least one fungal gene selected from the group consisting of MIC19, TO
  • Item 3 The yeast or filamentous fungal cell according to Item 1 or 2, wherein said genes or SUTs or CUTs are furthermore selected from the group of genes or SUTs or CUTs having a value of logFC/FDR of more than 200, more preferred of more than 300, and most preferred of more than 500, based on the values as determined herein.
  • Item 4 The yeast or filamentous fungal cell according to any one of Items 1 to 3, further comprising a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions.
  • Item 5 The yeast or filamentous fungal cell according to any one of Items 1 to 4, further comprising the fungal gene PDI1, showing an increased expression and/or overexpression.
  • yeast or filamentous fungal cell according to any one of Items 1 to 5, wherein said yeast or filamentous fungal cell is selected from the group consisting of Aspergillus spp., Trichoderma spp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces ssp., Pichia spp., Hansenula polymorpha, Fusarium spp., Neurospora spp., and Penicillium spp., preferably Saccharomyces cerevisiae.
  • Item 7 The yeast or filamentous fungal cell according to any one of Items 1 to 6, wherein said at least one secreted protein of interest also shows an increased expression and/or overexpression.
  • Item 8 The yeast or filamentous fungal cell according to any one of Items 1 to 7, wherein said at least one fungal gene showing increased expression and/or overexpression and/or showing reduced expression and/or inactivation is a native gene and/or is a recombinant gene, wherein preferably said recombinant gene is integrated into the genome as an expression cassette and/or extrachromosomally expressed, preferably using a replicative expression vector.
  • Item 9 The yeast or filamentous fungal cell according to any one of Items 1 to 8, wherein the cell furthermore comprises at least one additional recombinant secretion promoting gene, for example a gene for a chaperone, for a foldase and/or for a glycosylation-promoting protein.
  • additional recombinant secretion promoting gene for example a gene for a chaperone, for a foldase and/or for a glycosylation-promoting protein.
  • Item 10 The yeast or filamentous fungal cell according to any one of Items 1 to 9, wherein the increased expression and/or overexpression or reduced expression and/or inactivation of the at least one fungal gene or the at least one additional recombinant secretion promoting gene is constitutive or inducible.
  • Item 11 The yeast or filamentous fungal cell according to any one of Items 1 to 10, wherein the cell produces the at least one secreted protein to about 30% or more, or to about 40% or more, preferably about 50% or more, more preferably to about 75% or more, when compared to a control yeast or filamentous fungal cell.
  • Item 12 A method for producing a secreted protein in a yeast or filamentous fungal cell, comprising the steps of i) providing a yeast or filamentous fungal cell producing at least one secreted protein of interest according to any one of Items 1 to 11, ii) culturing said yeast or filamentous fungal cell in suitable culture medium, and iii) isolating said secreted protein from said culture medium.
  • Item 13 The method according to Item 12, further comprising suitably inducing the increased expression and/or overexpression or reduced expression and/or inactivation of the at least one fungal gene.
  • Item 14 The method according to Item 11 or 12, wherein about 30% or more, or about 40% or more, preferably about 50% or more, more preferably to about 75% or more of said at least one secreted protein is produced, when compared to the production of a control yeast or filamentous fungal cell.
  • a method for producing a yeast or filamentous fungal cell producing at least one secreted protein of interest comprising introducing into said cell producing at least one secreted protein of interest at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1, RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, and WBP1, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, NDC1, PET100, NIP7, VHT1, and SUT685, wherein said at least one fungal gene shows reduced expression and/or inactivation
  • Item 16 The method according to Item 15, further introducing into said cell a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions.
  • Item 17 The method according to Item 15 or 16, further introducing into said cell the fungal gene PDI1, showing an increased expression and/or overexpression.
  • Item 18 The method according to any one of Items 15 to 17, wherein said at least one fungal gene is integrated into the genome as an expression cassette and/or extrachromosomally expressed, preferably using a replicative expression vector.
  • Item 19 Use of a yeast or filamentous fungal cell according to any one of Items 1 to 10 for producing at least one secreted protein of interest.
  • Figure 1 shows the map of plasmid pLI410-062 as used in the methods according to the present invention.
  • Figure 2 A and B shows the results of the ⁇ -amylase secretion measurements relative to baseline for selected genes of the present invention as box plots in % control over time (4, 24, 48, and 120 hours).
  • Genes are ALP1, BNA7, GMH1, SUT074, TFG2, ENO2, NMA2, PRY2, and TOM22.
  • HAC1 is control.
  • Figure 3 A and B shows the results of the ⁇ -amylase secretion measurements per cell for selected genes of the present invention as box plots in % control over time (4, 24, 48, and 120 hours).
  • Genes are ALP1, BNA7, GMH1, SUT074, TFG2, ENO2, NMA2, PRY2, and TOM22.
  • HAC1 is control.
  • Figure 4 A and B shows the results of the ⁇ -amylase secretion measurements (total amylase) for selected genes of the present invention as box plots in % control over time (4, 24, and 48 hours).
  • Genes are INP51, MNT2, TLG2, TPO2, and YDR262W.
  • HAC1, HDA2 and ER.sec2 are controls.
  • Figure 5 A and B shows the results of the ⁇ -amylase secretion measurements per cell for selected genes of the present invention as box plots in % control overtime (4, 24, and 48 hours).
  • Genes are INP51, MNT2, TLG2, TPO2, and YDR262W.
  • HAC1, HDA2 and ER.sec2 are controls.
  • candidate genes were cloned into plasmid pLI410-062 between the Asc I and Sbf I restriction sites, which was then linearized by NotI enzyme, and transformed into yeast strain ER.sec2.
  • the plasmid integrates into the yeast chromosome at the BUD5 locus ( Figure 1 ).
  • native candidate genes were cloned into plasmid p427-TEF between SpeI and SalI and transformed into yeast strain ER.sec2.
  • Deletion strains were constructed by golden gate assembly of annealed oligos with gRNA sequences targeting the start and end position of the target gene, into sgRNA expression vector pWS082.
  • the assembled plasmid and Cas9 expression vector pWS173 were linearized using EcoRV or BsmBI and co-transformed with annealed repair fragments, consisting of the joined 60 bp flanking regions of each target gene, which upon successful homology directed repair, resulted in the deletion of the target gene in ER.sec2.
  • Preculture of YPD was performed, either with 22h of culture on SD-2xSCAA, or 22h and 96 h of culture on YPD.
  • SD-2 ⁇ SCAA medium was prepared as described previously (Hackel et al. 2006; Tyo et al.
  • SD-2xSCAA was as follows: 10 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids, 2 g/L, KH2PO4 (pH 6.0 by NaOH), and 1 g/L BSA, containing filter sterilized SCAA solution (190 mg/L arginine, 108 mg/L methionine, 52 mg/L tyrosine, 290 mg/L isoleucine, 440 mg/L lysine, 200 mg/L phenylalanine, 1,260 mg/L, glutamic acid, 400 mg/L aspartic acid, 380 mg/L valine, 220 mg/L threonine, 130 mg/L glycine, 400 mg/L leucine, 40 mg/L tryptophan, and 140 mg/L histidine) (see Liu et al., 2013 - Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae).
  • the initial OD 600nm was 0.1, and flasks of 250 ml + 50 ml of medium were used. Culture density was measured at OD 600nm .
  • E 400 Sample absorbance - blank absorbance, 10 : time of reaction, 0.17 : total volume of reaction, 0.01 : volume of sample, 18.1 : E mM p-nitrophenol in Trizma base 1%, D : Dilution of sample. Normalization of ⁇ -amylase activity was performed with respective OD 600nm .
  • logFC log fold change
  • FDR false discovery rate
  • logFC log fold change
  • FDR false discovery rate
  • logFC log fold change
  • FDR false discovery rate
  • Genes that were overexpressed - most preferred selection Gene (common name, SUT or CUT or systematic designation) Name and function (if known) logFC FDR MIC19 Component of the MICOS complex 13.883 0.036 TOM22 Translocase of the Outer Mitochondrial membrane; responsible for initial import of mitochondrially directed proteins 13.781 0.008 NKP1 Non-essential Kinetochore Protein 13.389 0.012 DML1 Drosophila melanogaster Misato-Like protein, Essential protein involved in mtDNA inheritance 13.307 0.014 CUT859 SUT or CUT 13.152 0.033 GAL80 GALactose metabolism, Transcriptional regulator involved in the repression of GAL genes 12.170 0.008 APM3 clathrin Adaptor Protein complex Medium chain 12.088 0.020 COQ10 COenzyme Q, Coenzyme Q (ubiquinone) binding protein 12.048 0.025 BLM10 BLeoMycin resistance, Proteasome activ
  • genes or SUTs or CUTs that are selected from the group of genes or SUTs or CUTs having a value of logFC/FDR of more than 200, more preferred of more than 300, and most preferred of more than 500, based on the values herein.

Abstract

The present invention relates to a yeast or filamentous fungal cell producing at least one secreted protein of interest, wherein said cell comprises at least one additional fungal gene showing increased expression and/or overexpression, showing reduced expression and/or inactivation, wherein said gene improves the production of the at least one secreted protein of interest. The present invention further relates to respective methods for production and uses of the yeast or filamentous fungal cells.

Description

  • The present invention relates to a yeast or filamentous fungal cell producing at least one secreted protein of interest, wherein said cell comprises at least one additional fungal gene showing increased expression and/or overexpression, showing reduced expression and/or inactivation, wherein said gene improves the production of the at least one secreted protein of interest. The present invention further relates to respective methods for production and uses of the yeast or filamentous fungal cells.
    • Background of the invention
  • The production of recombinant enzymes is growing rapidly and is estimated to generate several tens of billions of dollars (Martinez et al., 2012). Almost 60% of the enzymes used in detergents, the food industry and biofuel alcohol are recombinant enzymes, i.e. produced by an organism other than that of origin of the protein (COWAN, 1996). The expression of enzymes in a heterologous host allows (i) the production of enzymes of interest from slow growing or even non-cultivable organisms, (ii) the much higher production of the enzyme of interest, (iii ) the production of proteins from pathogenic or toxin-producing organisms, and (iv) the increase of the stability or activity of an enzyme by protein engineering (Falch, 1991; Demain and Vaishnav, 2009).
  • Many microorganisms, including filamentous fungi (Aspergillus sp., Trichoderma sp.), yeasts (for example Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica) or bacteria (for example Escherichia coli, Bacillus sp.), are used to produce recombinant proteins (Demain and Vaishnav, 2009).
  • The production of recombinant proteins is dependent on the expression cassette (promoters and terminators used, signal sequence, codon bias), on the cellular machinery involved in the synthesis and degradation of proteins, intracellular trafficking and/or secretion, but also the energy level and/or redox of the cell as well as the culture conditions and the availability of nutrients (Zahrl et al., 2019).
  • Compared to other organisms conventionally used to produce recombinant proteins, S. cerevisiae has the advantage of rapid growth, easy manipulation both at the genetic level and at the level of production in bioreactors, and having Generally Recognized As Safe (GRAS) status. The production of a heterologous target protein in yeast host cells is further advantageous in that it allows the target proteins to be folded and secreted through the cellular secretory machinery.
  • Yeast is already widely used for many industrial applications (breadmaking, production of drinking alcohol and biofuels, etc. Parapouli et al., 2020) where it may be advantageous to have it produce heterologous enzymes. For example, in the field of biofuel alcohol, the commercialized yeast strains of S. cerevisiae secrete enzymatic activities allowing the degradation of industrial mashes containing starch derivatives. This allows bioethanol manufacturers to limit their intake of exogenous enzymes and reduce their production costs.
  • US 2011-0129872A1 relates to a method for producing a recombinant protein, comprising culturing a yeast transformed with a recombinant gene construct comprising a yeast promoter, a gene coding a signal sequence and a gene coding a target protein; and also with one or more genes coding folding accessory protein selected from the group consisting of PDI1 (protein disulfide isomerase 1), SEC23 (secretory 23), TRX2 (thioredoxin 2) AHA1 (activator of heat shock protein 90 ATPase), and SCJ1 (S. cerevisiae DnaJ), followed by culturing the transformed yeast.
  • US 2013-0011875 relates to a method and the production of higher titers of recombinant protein in a modified yeast host cell, for example Pichia pastoris, wherein the modified yeast cell lacks vacuolar sorting activity or has decreased vacuolar sorting activity relative to an unmodified yeast host cell of the same species.
  • US 2014-0335622 discloses an expression vector for secreting a protein (Z) to be recovered or a fusion protein having the protein (Z) moiety therein; a method for producing a transformant using the expression vector; the transformant; and a method for producing a protein using the transformant. It is disclosed that co-expression of a foreign secretory protein with PDI1 increases the secretory production amount.
  • US 2016-0186192 describes a method for producing a desired protein comprising: (a) providing a host cell comprising a first recombinant gene encoding a protein comprising the sequence of a first chaperone protein, a second recombinant gene encoding a protein comprising the sequence of a second chaperone protein and a third gene, such as a third recombinant gene, encoding a desired protein (such as a desired heterologous protein), wherein the first and second chaperones are different; and (b) culturing the host cell in a culture medium to obtain expression of the first, second and third genes.
  • US 2018-0022785 claims a method for producing a heterologous protein, said method comprising: culturing a Saccharomyces cerevisiae yeast host cell or a culture thereof to produce the heterologous protein, wherein said Saccharomyces cerevisiae yeast host cell comprises a modified Not4 protein, and wherein said heterologous protein is an albumin, or a variant, fragment and/or fusion thereof.
  • Eun Jung Thak et al. (in: Yeast synthetic biology for designed cell factories producing secretory recombinant proteins, FEMS Yeast Research, Volume 20, Issue 2, March 2020, foaa009, https://doi.org/10.1093/femsyr/foaa009) disclose that yeasts are prominent hosts for the production of recombinant proteins from industrial enzymes to therapeutic proteins. Particularly, the similarity of protein secretion pathways between these unicellular eukaryotic microorganisms and higher eukaryotic organisms has made them a preferential host to produce secretory recombinant proteins. However, there are several bottlenecks, in terms of quality and quantity, restricting their use as secretory recombinant protein production hosts. They discuss recent developments in synthetic biology approaches to constructing yeast cell factories endowed with enhanced capacities of protein folding and secretion as well as designed targeted post-translational modification process functions, and focus on the new genetic tools for optimizing secretory protein expression, such as codon-optimized synthetic genes, combinatory synthetic signal peptides and copy number-controllable integration systems, and the advanced cellular engineering strategies, including endoplasmic reticulum and protein trafficking pathway engineering, synthetic glycosylation, and cell wall engineering, for improving the quality and yield of secretory recombinant proteins.
  • Zihe Liu, etal. (in: Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering, Applied and Environmental Microbiology Aug 2014, 80 (17) 5542-5550; DOI: 10.1128/AEM.00712-14) disclose that the increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. They applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in the VTA1 gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.
  • Huang, M., et al. (in: Efficient protein production by yeast requires global tuning of metabolism. Nat Commun 8, 1131 (2017). https://doi.org/10.1038/s41467-017-00999-2) describe that the biotech industry relies on cell factories for production of pharmaceutical proteins, of which several are among the top-selling medicines. There is, therefore, considerable interest in improving the efficiency of protein production by cell factories. Protein secretion involves numerous intracellular processes with many underlying mechanisms still remaining unclear. They used RNA-seq to study the genome-wide transcriptional response to protein secretion in mutant yeast strains, and find that many cellular processes have to be attuned to support efficient protein secretion. In particular, altered energy metabolism resulting in reduced respiration and increased fermentation, as well as balancing of amino-acid biosynthesis and reduced thiamine biosynthesis seem to be particularly important. They confirmed their findings by inverse engineering and physiological characterization and show that by tuning metabolism cells are able to efficiently secrete recombinant proteins.
  • Huang M, et al. (In: Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast. Proc Natl Acad Sci U S A. 2015 Aug 25;112(34):E4689-96. doi: 10.1073/pnas.1506460112. Epub 2015 Aug 10. PMID: 26261321; PMCID: PMC4553813) disclose that there is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications, that the yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is said to be difficult to apply rational engineering for construction of improved strains. They used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. They performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that had not been identified before in the context of protein secretion. Mutated genes identified are disclosed to be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.
  • Finally, Huang M, et al. (in: Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production. Proc Natl Acad Sci U S A. 2018 Nov 20;115(47):E11025-E11032. doi: 10.1073/pnas.1809921115. Epub 2018 Nov 5. PMID: 30397111; PMCID: PMC6255153) describe that baker's yeast Saccharomyces cerevisiae is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. They identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications of endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, they enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.
  • Cryptic unstable transcripts (CUTs) are a subset of non-coding RNAs (ncRNAs) that are produced from intergenic and intragenic regions. Additionally, stable uncharacterized transcripts, or SUTs, have also been detected in cells and bear many similarities to CUTs but are not degraded through the same pathways.
  • Genetic engineering strategies to overcome bottlenecks in the yeast protein secretion pathway have to consider that protein secretion in yeast involves multiple complex steps, such as protein translocation, folding, post-translational modification and vesicle trafficking between several membrane organelles and plasma membranes. The secretion of proteins synthesized inside cells can be hampered by low secretion efficiency, abnormal post-translational modifications, retention within the secretion pathway or the cell wall space as a cell-associated form. The development of engineering strategies targeted to each step of the secretion pathway in a modular fashion is required in order to design cell factories producing secretory recombinant proteins. Today, despite its obvious qualities, S. cerevisiae remains relatively limited in its ability to secrete proteins compared to organisms such as filamentous fungi or P. pastoris (Demain and Vaishnav, 2009). It is therefore an object of the present invention to provide new factors to improve recombinant protein production and secretion in yeast. Other objects and advantages will become apparent to the person of skill when studying the present description of the present invention.
  • In a first aspect of the present invention, the above object is solved by providing a yeast or filamentous fungal cell producing at least one secreted protein of interest, wherein said cell comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, VHS2, ASA1, TRP4, YPS7, CUT824, YOR318C, PRM7, ERV46, FIT2, GPM3, CUT892, SRN2, SUT643, CUT461, THR4, GMH1, SOL1, NAB6, YPR148C, ALP1, CUT097, ATG33, YOR316C-A, SOG2, MCM6, SUT230, SUT419, TIF11, TAF5, PHO91, AIM32, ENO2, UBA2, PUS5, ERG1, SUT311, KSS1, MRP10, CUT598, CUT188, YOR238W, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1 RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, WBP1, AVT2, CUT854, TRM10, SLX9, YPL077C, PET122, TFG2, PUN1, CUT152, AIR2, CUT571, RPS26B, RRT6, RPC19, URA3, YGR045C, SMC3, PNG1, THI6, MEU1, CUT239, NSE4, SUT074, AAH1, RMD5, CUT607, ACS1, MNN1, ARH1, YHR140W, CET1, RRB1, YLR342W-A, RPS22B, CHS5, YIL165C, SUT093, LPX1, NCA3, EFG1, NBP35, CUT765, MSL1, SCD6, ATG42, CHS6, COQ2, RPO31, MKK1, HED1, PBP2, BET5, CUT678, YGR021W, SUT474, YGL159W, IRC21, VHR1, SPP1, PRP43, ZRT1, YLR041W, SUT711, COX18, CBP6, SUT575, CLG1, CUT213, QCR10, SNR3, MSS2, CUT505, YOS1, SUT073, UTP21, ACA1, CUT632, RIP1, HUL5, CUT727, RPL35B, CUT184, CUT420, YFL041W-A, SUT460, ATG10, MFA1, UGX2, TRK2, CUT704, SUT083, TRE1, RVS161, LEA1, EBP2, THI80, CTI6, CUT322, XPT1, MRPL35, YPL025C, SUT737, PGA2, ULP2, MRX16, EST1, NUP100, IES3, ATG39, YMR084W, SUT428, YPL119C-A, MIN8, CUT490, SUT287, KEL3, SUT678, SEC3, SOL4, SIS2, CUT915, RRP3, ESA1, PCL8, TRX3, YKL115C, EMP65, ZDS1, CUT167, SOD1, UBR2, LSP1, SNR81, RGD3, YTP1, SMY2, CUT449, FIN1, YKL106C-A, YAR019W-A, CCH1, AYR1, SUT573, VNX1, FOL3, SUT511, GIS4, CUT743, RPL24A, HMT1, SUT333, SPP2, SUT128, SMC6, PHR1, RPS15, CUT642, GYP7, tK(CUU)K, CUT896, SLM5, CUT586, CUT158, and RRP12, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, YDR262W, CMC1, MRP17, YPT52, CUT312, MRPS5, RDR1, DAL7, RPL20A, YBR137W, RPL36B, YEL008C-A, RAX1, CUT729, INP51, UBP8, CUT258, YLR342W-A, SUT568, PEX7, MSD1, CUT136, TIM10, CUT361, snR51, TAL1, RIP1, MRP10, SUT078, MRP51, GLO3, EHD3, HER1, NMA111, PBP4, MFB1, IKI3, NDL1, SUT433, YOR238W, SUT750, QDR2, RDI1, SUT014, CUT437, MSC6, SUT497, YCR051W, MRPL33, RPL14A, TRM7, RNH202, RTC5, SUT027, CDC5, SUT729, YOR131C, CUT665, GLG2, SUT268, SUT705, MED4, RCR2, EFB1, RXT2, KGD1, TUP1, RNH203, YDR338C, SED1, CUT522, HIS2, SUT145, MET17, APC4, NKP2, MKK2, NDC1, PET100, NIP7, VHT1, SUT685, BNI5, SNA3, EGH1, MRP4, POB3, PIB2, SUT317, and NTO1, wherein said at least one fungal gene shows reduced expression and/or inactivation.
  • Preferably, said cell comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1, RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, and WBP1, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, NDC1, PET100, NIP7, VHT1, and SUT685, wherein said at least one fungal gene shows reduced expression and/or inactivation..
  • More preferred is the yeast or filamentous fungal cell according to the present invention, wherein said genes or SUTs or CUTs are furthermore selected from the group of genes or SUTs or CUTs having a value of logFC/FDR of more than 200, more preferred of more than 300, and most preferred of more than 500, based on the values as determined herein.
  • More preferred is the yeast or filamentous fungal cell according to the present invention, further comprising a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions, such as, without wanting to be bound by theory, for example, the impact of CRISPRa and CRISPRi on gene expression due to the position of the gRNA in the promoting region.
  • Even more preferred is the yeast or filamentous fungal cell according to the present invention, further comprising the fungal gene PDI1, showing an increased expression and/or overexpression.
  • Advantageously, the yeast or filamentous fungal cell according to the present invention produces the at least one secreted protein to about 30% or more, or about 40% or more, preferably about 50% or more, more preferably to about 75% or more, when compared to a control yeast or filamentous fungal cell.
  • In a second aspect of the present invention, the above object is solved by a method for producing a secreted protein in a yeast or filamentous fungal cell, comprising the steps of i) providing a yeast or filamentous fungal cell producing at least one secreted protein of interest according to the present invention, ii) culturing said yeast or filamentous fungal cell in suitable culture medium, and iii) isolating said secreted protein from aid culture medium. Preferred is the method according to the present invention, further comprising suitably inducing the increased expression and/or overexpression or reduced expression and/or inactivation of the at least one fungal gene.
  • Further preferred is the method according to the present invention, wherein about 30% or more, or about 40% or more, preferably about 50% or more, more preferably to about 75% or more of said at least one secreted protein is produced, when compared to the production of a control yeast or filamentous fungal cell.
  • In a third aspect of the present invention, the above object is solved by a method for producing a yeast or filamentous fungal cell producing at least one secreted protein of interest according to the present invention, comprising introducing into said cell producing at least one secreted protein of interest at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, VHS2, ASA1, TRP4, YPS7, CUT824, YOR318C, PRM7, ERV46, FIT2, GPM3, CUT892, SRN2, SUT643, CUT461, THR4, GMH1, SOL1, NAB6, YPR148C, ALP1, CUT097, ATG33, YOR316C-A, SOG2, MCM6, SUT230, SUT419, TIF11, TAF5, PHO91, AIM32, ENO2, UBA2, PUS5, ERG1, SUT311, KSS1, MRP10, CUT598, CUT188, YOR238W, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1 RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, WBP1, AVT2, CUT854, TRM10, SLX9, YPL077C, PET122, TFG2, PUN1, CUT152, AIR2, CUT571, RPS26B, RRT6, RPC19, URA3, YGR045C, SMC3, PNG1, THI6, MEU1, CUT239, NSE4, SUT074, AAH1, RMD5, CUT607, ACS1, MNN1, ARH1, YHR140W, CET1, RRB1, YLR342W-A, RPS22B, CHS5, YIL165C, SUT093, LPX1, NCA3, EFG1, NBP35, CUT765, MSL1, SCD6, ATG42, CHS6, COQ2, RPO31, MKK1, HED1, PBP2, BET5, CUT678, YGR021W, SUT474, YGL159W, IRC21, VHR1, SPP1, PRP43, ZRT1, YLR041W, SUT711, COX18, CBP6, SUT575, CLG1, CUT213, QCR10, SNR3, MSS2, CUT505, YOS1, SUT073, UTP21, ACA1, CUT632, RIP1, HUL5, CUT727, RPL35B, CUT184, CUT420, YFL041W-A, SUT460, ATG10, MFA1, UGX2, TRK2, CUT704, SUT083, TRE1, RVS161, LEA1, EBP2, THI80, CTI6, CUT322, XPT1, MRPL35, YPL025C, SUT737, PGA2, ULP2, MRX16, EST1, NUP100, IES3, ATG39, YMR084W, SUT428, YPL119C-A, MIN8, CUT490, SUT287, KEL3, SUT678, SEC3, SOL4, SIS2, CUT915, RRP3, ESA1, PCL8, TRX3, YKL115C, EMP65, ZDS1, CUT167, SOD1, UBR2, LSP1, SNR81, RGD3, YTP1, SMY2, CUT449, FIN1, YKL106C-A, YAR019W-A, CCH1, AYR1, SUT573, VNX1, FOL3, SUT511, GIS4, CUT743, RPL24A, HMT1, SUT333, SPP2, SUT128, SMC6, PHR1, RPS15, CUT642, GYP7, tK(CUU)K, CUT896, SLM5, CUT586, CUT158, and RRP12, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, YDR262W, CMC1, MRP17, YPT52, CUT312, MRPS5, RDR1, DAL7, RPL20A, YBR137W, RPL36B, YEL008C-A, RAX1, INP51, CUT729, UBP8, CUT258, YLR342W-A, SUT568, PEX7, MSD1, CUT136, TIM10, CUT361, snR51, TAL1, RIP1, MRP10, SUT078, MRP51, GLO3, EHD3, HER1, NMA111, PBP4, MFB1, IKI3, NDL1, SUT433, YOR238W, SUT750, QDR2, RDI1, SUT014, CUT437, MSC6, SUT497, YCR051W, MRPL33, RPL14A, TRM7, RNH202, RTC5, SUT027, CDC5, SUT729, YOR131C, CUT665, GLG2, SUT268, SUT705, MED4, RCR2, EFB1, RXT2, KGD1, TUP1, RNH203, YDR338C, SED1, CUT522, HIS2, SUT145, MET17, APC4, NKP2, MKK2, NDC1, PET100, NIP7, VHT1, SUT685, BNI5, SNA3, EGH1, MRP4, POB3, PIB2, SUT317, and NTO1, wherein said at least one fungal gene shows reduced expression and/or inactivation..
  • Preferred is a method of the present invention for producing a yeast or filamentous fungal cell producing at least one secreted protein of interest according to the present invention, comprising introducing into said cell producing at least one secreted protein of interest at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1, RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, and WBP1, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, NDC1, PET100, NIP7, VHT1, and SUT685, wherein said at least one fungal gene shows reduced expression and/or inactivation.
  • Furthermore, the method according to the invention may include further introducing into said cell a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions. Furthermore, the method may include further introducing into said cell the fungal gene PDI1, showing an increased expression and/or overexpression.
  • In a fourth aspect of the present invention, the above object is solved by the use of a yeast or filamentous fungal cell according to the present invention for producing at least one secreted protein of interest.
  • As mentioned above, the analysis of UV S. cerevisiae mutants expressing an α-amylase has revealed improved strains for secretion (Huang et al., 2015; Huang et al., 2018). Coupling microfluidics with a phenotypic screening using a starch complexed with BODIPY (which becomes fluorescent when it is released), the authors had selected the mutants secreting the most enzyme into the extracellular medium. The sequencing of eight hypersecretory clones (x1.5 to x6) revealed 330 mutations potentially involved in improving α-amylase production and secretion (Huang et al., 2015). A more in-depth analysis led to the identification of - amongst others as disclosed herein - a role of the known PDI1 gene in the production and secretion of α-amylase in S. cerevisiae.
  • The purpose of the present invention was to discover new factors and genes involved in protein secretion in order to improve protein production and secretion, as exemplified in the industrial Ethanol Red ® strain of S. cerevisiae.
  • As mentioned above, in the first aspect of the present invention, a yeast or filamentous fungal cell is provided that produces at least one secreted protein of interest. In addition, the cell comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, VHS2, ASA1, TRP4, YPS7, CUT824, YOR318C, PRM7, ERV46, FIT2, GPM3, CUT892, SRN2, SUT643, CUT461, THR4, GMH1, SOL1, NAB6, YPR148C, ALP1, CUT097, ATG33, YOR316C-A, SOG2, MCM6, SUT230, SUT419, TIF11, TAF5, PHO91, AIM32, ENO2, UBA2, PUS5, ERG1, SUT311, KSS1, MRP10, CUT598, CUT188, YOR238W, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1 RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, WBP1, AVT2, CUT854, TRM10, SLX9, YPL077C, PET122, TFG2, PUN1, CUT152, AIR2, CUT571, RPS26B, RRT6, RPC19, URA3, YGR045C, SMC3, PNG1, THI6, MEU1, CUT239, NSE4, SUT074, AAH1, RMD5, CUT607, ACS1, MNN1, ARH1, YHR140W, CET1, RRB1, YLR342W-A, RPS22B, CHS5, YIL165C, SUT093, LPX1, NCA3, EFG1, NBP35, CUT765, MSL1, SCD6, ATG42, CHS6, COQ2, RPO31, MKK1, HED1, PBP2, BET5, CUT678, YGR021W, SUT474, YGL159W, IRC21, VHR1, SPP1, PRP43, ZRT1, YLR041W, SUT711, COX18, CBP6, SUT575, CLG1, CUT213, QCR10, SNR3, MSS2, CUT505, YOS1, SUT073, UTP21, ACA1, CUT632, RIP1, HUL5, CUT727, RPL35B, CUT184, CUT420, YFL041W-A, SUT460, ATG10, MFA1, UGX2, TRK2, CUT704, SUT083, TRE1, RVS161, LEA1, EBP2, THI80, CTI6, CUT322, XPT1, MRPL35, YPL025C, SUT737, PGA2, ULP2, MRX16, EST1, NUP100, IES3, ATG39, YMR084W, SUT428, YPL119C-A, MIN8, CUT490, SUT287, KEL3, SUT678, SEC3, SOL4, SIS2, CUT915, RRP3, ESA1, PCL8, TRX3, YKL115C, EMP65, ZDS1, CUT167, SOD1, UBR2, LSP1, SNR81, RGD3, YTP1, SMY2, CUT449, FIN1, YKL106C-A, YAR019W-A, CCH1, AYR1, SUT573, VNX1, FOL3, SUT511, GIS4, CUT743, RPL24A, HMT1, SUT333, SPP2, SUT128, SMC6, PHR1, RPS15, CUT642, GYP7, tK(CUU)K, CUT896, SLM5, CUT586, CUT158, and RRP12, wherein these at least one fungal gene shows increased expression and/or overexpression. In the context of the present invention, the terms "increased expression" or "overexpression" indicate that the amount of protein as produced by the cell is higher when compared to the expression in a control cell showing normal, unaltered or baseline expression. The change in expression can be achieved in any suitable way, and examples include mutated promotors, cloning of the gene under the control of a heterologous "strong" promotor, either inducible or constitutive, codon optimization, and mutations that stabilize the structure of the protein, and the like. Alternatively or in addition, the cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, YDR262W, CMC1, MRP17, YPT52, CUT312, MRPS5, RDR1, DAL7, RPL20A, YBR137W, RPL36B, YEL008C-A, RAX1, INP51, CUT729, UBP8, CUT258, YLR342W-A, SUT568, PEX7, MSD1, CUT136, TIM10, CUT361, snR51, TAL1, RIP1, MRP10, SUT078, MRP51, GLO3, EHD3, HER1, NMA111, PBP4, MFB1, IKI3, NDL1, SUT433, YOR238W, SUT750, QDR2, RDI1, SUT014, CUT437, MSC6, SUT497, YCR051W, MRPL33, RPL14A, TRM7, RNH202, RTC5, SUT027, CDC5, SUT729, YOR131C, CUT665, GLG2, SUT268, SUT705, MED4, RCR2, EFB1, RXT2, KGD1, TUP1, RNH203, YDR338C, SED1, CUT522, HIS2, SUT145, MET17, APC4, NKP2, MKK2, NDC1, PET100, NIP7, VHT1, SUT685, BNI5, SNA3, EGH1, MRP4, POB3, PIB2, SUT317, and NTO1, wherein said at least one fungal gene shows reduced expression and/or inactivation, wherein said at least one fungal gene shows reduced expression and/or inactivation. In the context of the present invention, the terms "reduced expression" or "inactivation" indicate that the amount of protein as produced by the cell is lower when compared to the expression in a control cell showing normal, unaltered or baseline expression. The change in expression can be achieved in any suitable way, and examples include mutated promotors, cloning of the gene under the control of a heterologous "weak" promotor, either inducible or constitutive, codon changes, and mutations that de-stabilize the structure of the protein, and the like.
  • Preferably, said yeast or filamentous fungal cell as provided comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1, RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, and WBP1, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, NDC1, PET100, NIP7, VHT1, and SUT685, wherein said at least one fungal gene shows reduced expression and/or inactivation.
  • More preferred is the yeast or filamentous fungal cell according to the present invention, wherein said genes or SUTs or CUTs are furthermore selected from the group of genes or SUTs or CUTs having a value of logFC/FDR of more than 200, more preferred of more than 300, and most preferred of more than 500, based on the values as determined herein.
  • More preferred is the yeast or filamentous fungal cell according to the present invention, further comprising a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions.
  • Even more preferred is the yeast or filamentous fungal cell according to the present invention, further comprising the fungal gene PDI1, showing an increased expression and/or overexpression.
  • It is expected that a combination of genes as mentioned herein can lead to an even further increased production of the protein of interest, even having synergistic effects. Examples for these combinations are all of TLG2, YDR262W, and TRM10, optionally further comprising PDI1. Other examples are ATG33 and MRP10, NDC1 and TRM10, or PRY2, and TOM22, again each pair optionally further comprising PDI1.
  • Most preferred are either AVT2, PRY2, SUT074, BNA7, TOM22 or TRM10. The overexpression of AVT2, TRM10, PRY2, SUT074, BNA7, or TOM22, and the inactivation of INP51 is further preferred. Further examples are TLG2, CUT901, ATG33, THR4, YDR262W, and CMC1, optionally further comprising PDI1.
  • The fungal gene(s) and/or SUTs or CUTs as used are preferably derived from S. cerevisiae, or a related yeast. The fungal gene(s) and/or SUTs or CUTs and their reference numbers are according to the Saccharomyces Genome Database (SGD) (https://www.yeastgenome.org/), as of November 15, 2021. Related genes that may be used as well encode for proteins sharing the same biological effect (increased secretion) in the yeast or filamentous fungal cell with the genes as above, and/or have an amino acid identity of about 80% or more, preferably about 90% or more, more preferably about 95% or more with the polypeptide sequence as encoded by a genes as above.
  • Advantageously, preferably the yeast or filamentous fungal cell according to the present invention produces the at least one secreted protein to about 30% or more or 40% or more, preferably about 50% or more, more preferably to about 75% or more, when compared to a control yeast or filamentous fungal cell, preferably one that does not contain a gene as mentioned above leading to increased secretion of the protein of interest.
  • As the protein of interest, any protein can be chosen that can be suitably produced by the yeast or filamentous fungal cell according to the present invention, e.g. expressed, folded, glycosylated and/or secreted. The gene of the protein of interest can be codon optimized, and preferably show an increased expression and/or overexpression, as explained above for the fungal gene according to the present invention. Examples of preferred proteins of interest are human serum albumin (HSA), amylase, human insulin, and components of hepatitis vaccines, human papillomavirus (HPV) vaccines, interferon(s), or epidermal growth factor (hEGF), and proteins used in food production, such as cellulase, glucoamylase, xylanase, and the like.
  • In order to identify new genes involved in the production and secretion of recombinant and heterologous proteins in yeast or filamentous fungal cells, such as S. cerevisiae, the inventors have developed CRISPRi and CRISPRa libraries allowing the overexpression or the repression of all genes as well as previously identified Stable Unannotated Transcripts (SUT's) and (Cryptic Unstable Transcripts CUT's) of this yeast (see Xu, Z. et al. Bidirectional promoters generate pervasive transcription in yeast. Nature 457, 1033-1037 (2009)). These libraries utilize an inactivated Cas9 (dCas9) able to bind DNA at the CRISPR site but unable to cleave the DNA molecule, fused to a transcriptional activation (CRISPRa) (e.g. the VP64-p65-Rta (VPR) tripartite activation domain described in Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12, 326-328 (2015)) or repression domain (CRISPRi) (Dominguez et al., 2015).
  • The industrial Ethanol Red® (ER) yeast strain overexpressing an α-amylase (Amy6 from A. niger) was used as a model for the present invention (Lesaffre, Marcq-en-Barœul, France). A 40,890 gRNA library targeting the promoters of 7,247 yeast genes, SUT's and CUT's at an average of 5.8 positions per gene, SUT or CUT was developed and cloned into replicative vectors allowing their expression as well as the expression of dCas9-VP64-p65-Rta (CRISPRa) or dCas9-Mxi1 (CRISPRi). The ER + α-amylase strain was then transformed using the CRISPRa and CRISPRi libraries, and the cell population as obtained was screened by microfluidics on the basis of its capacity to degrade a starch substrate labelled with BODIPY FL dye which fluoresces in green when the starch is degraded by α-amylase (e.g. EnzChek® Ultra Amylase Assay Kit: https://www.thermofisher.com/order/catalog/product/E33651#/E33651).
  • Clones presenting high fluorescence were sorted, and gRNA regions from replicative vectors were analyzed by Illumina sequencing. Data analysis revealed that 320 activated or repressed genes favor α-amylase secretion. These genes were manually selected further, and the genes MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, VHS2, ASA1, TRP4, YPS7, CUT824, YOR318C, PRM7, ERV46, FIT2, GPM3, CUT892, SRN2, SUT643, CUT461, THR4, GMH1, SOL1, NAB6, YPR148C, ALP1, CUT097, ATG33, YOR316C-A, SOG2, MCM6, SUT230, SUT419, TIF11, TAF5, PHO91, AIM32, ENO2, UBA2, PUS5, ERG1, SUT311, KSS1, MRP10, CUT598, CUT188, YOR238W, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1 RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, WBP1, AVT2, CUT854, TRM10, SLX9, YPL077C, PET122, TFG2, PUN1, CUT152, AIR2, CUT571, RPS26B, RRT6, RPC19, URA3, YGR045C, SMC3, PNG1, THI6, MEU1, CUT239, NSE4, SUT074, AAH1, RMD5, CUT607, ACS1, MNN1, ARH1, YHR140W, CET1, RRB1, YLR342W-A, RPS22B, CHS5, YIL165C, SUT093, LPX1, NCA3, EFG1, NBP35, CUT765, MSL1, SCD6, ATG42, CHS6, COQ2, RPO31, MKK1, HED1, PBP2, BET5, CUT678, YGR021W, SUT474, YGL159W, IRC21, VHR1, SPP1, PRP43, ZRT1, YLR041W, SUT711, COX18, CBP6, SUT575, CLG1, CUT213, QCR10, SNR3, MSS2, CUT505, YOS1, SUT073, UTP21, ACA1, CUT632, RIP1, HUL5, CUT727, RPL35B, CUT184, CUT420, YFL041W-A, SUT460, ATG10, MFA1, UGX2, TRK2, CUT704, SUT083, TRE1, RVS161, LEA1, EBP2, THI80, CTI6, CUT322, XPT1, MRPL35, YPL025C, SUT737, PGA2, ULP2, MRX16, EST1, NUP100, IES3, ATG39, YMR084W, SUT428, YPL119C-A, MIN8, CUT490, SUT287, KEL3, SUT678, SEC3, SOL4, SIS2, CUT915, RRP3, ESA1, PCL8, TRX3, YKL115C, EMP65, ZDS1, CUT167, SOD1, UBR2, LSP1, SNR81, RGD3, YTP1, SMY2, CUT449, FIN1, YKL106C-A, YAR019W-A, CCH1, AYR1, SUT573, VNX1, FOL3, SUT511, GIS4, CUT743, RPL24A, HMT1, SUT333, SPP2, SUT128, SMC6, PHR1, RPS15, CUT642, GYP7, tK(CUU)K, CUT896, SLM5, CUT586, CUT158, and RRP12, were overexpressed using common techniques (integration of overexpression cassette into the genome and/or overexpression through a replicative plasmid), and genes TLG2, CUT901, ATG33, THR4, YDR262W, CMC1, MRP17, YPT52, CUT312, MRPS5, RDR1, DAL7, RPL20A, YBR137W, RPL36B, YEL008C-A, RAX1, INP51, CUT729, UBP8, CUT258, YLR342W-A, SUT568, PEX7, MSD1, CUT136, TIM10, CUT361, snR51, TAL1, RIP1, MRP10, SUT078, MRP51, GLO3, EHD3, HER1, NMA111, PBP4, MFB1, IKI3, NDL1, SUT433, YOR238W, SUT750, QDR2, RDI1, SUT014, CUT437, MSC6, SUT497, YCR051W, MRPL33, RPL14A, TRM7, RNH202, RTC5, SUT027, CDC5, SUT729, YOR131C, CUT665, GLG2, SUT268, SUT705, MED4, RCR2, EFB1, RXT2, KGD1, TUP1, RNH203, YDR338C, SED1, CUT522, HIS2, SUT145, MET17, APC4, NKP2, MKK2, NDC1, PET100, NIP7, VHT1, SUT685, BNI5, SNA3, EGH1, MRP4, POB3, PIB2, SUT317, and NTO1, were inactivated by gene deletion. Then, α-amylase activity was evaluated in the respective strains. The overexpression of BNA7, SUT074, TOM22, TLG2, YDR262W, ALP1, ENO2, NMA2, PRY2, and INP51 were identified as preferred for the exemplary α-amylase secretion in the Ethanol Red® strain.
  • In the context of the present invention, any suitable cell of a yeast or filamentous fungus can be used for the production of the protein of interest according to the present invention. Preferably, said yeast or filamentous fungal cell is selected from the group consisting of Aspergillus spp., Trichoderma spp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces ssp., Pichia spp., Hansenula polymorpha, Fusarium spp., Neurospora spp., and Penicillium spp., preferably Saccharomyces cerevisiae.
  • In the yeast or filamentous fungal cell according to the present invention, the at least one fungal gene showing increased expression and/or overexpression and/or showing reduced expression and/or inactivation is a native gene and/or is a recombinant gene, i.e. a modified gene of the yeast or filamentous fungal cell itself, or at least one gene that is recombinantly introduced and may be a heterologous gene, i.e. coming from a different strain or fungal species. Preferably, the recombinant gene is integrated into the genome as an expression cassette. Respective expression cassettes for fungal expression are known, and basically consist of a promoter, the fungal gene, and a terminator. Alternatively or in additionally, the gene can be extrachromosomally expressed, preferably using a replicative expression vector, such as a shuttle vector. Promoters used in yeast and fungal expression systems are usually either inducible or constitutive.
  • The folding and glycosylation of the secretory proteins in the endoplasmatic reticulum (ENDR) is assisted by numerous ENDR-resident proteins. The chaperones like Bip (GRP78), GRP94 or yeast Lhs1p help the secretory protein to fold by binding to exposed hydrophobic regions in the unfolded states and preventing unfavourable interactions (Blond-Elguindi et al., 1993, Cell 75:717-728). The chaperones are also important for the translocation of the proteins through the ENDR membrane. The proteins like protein disulphide isomerase and its homologs and prolyl-peptidyl cis-trans isomerase assist in formation of disulphide bridges and formation of the right conformation of the peptide chain adjacent to proline residues, respectively. A machinery including many protein components also resides in the ENDR for the addition of the N-linked core glycans to the secretory protein and for the initial trimming steps of the glycans.
  • Preferred is therefore the yeast or filamentous fungal cell according to the present invention, wherein the cell furthermore comprises at least one additional recombinant secretion promoting gene, for example a fungal gene for a chaperone, for a foldase and/or for a glycosylation-promoting protein. Like the other genes as disclosed herein, these proteins may be controllably expressed, inducible, constitutive, and even overexpressed.
  • Therefore, preferred is the yeast or filamentous fungal cell according to the present invention, wherein the increased expression and/or overexpression or reduced expression and/or inactivation of the at least one fungal gene or the at least one additional recombinant secretion promoting gene is constitutive or inducible.
  • Another important aspect of the present invention relates to a method for producing a secreted protein in a yeast or filamentous fungal cell, comprising the steps of i) providing a yeast or filamentous fungal cell producing at least one secreted protein of interest according to the present invention as above, ii) suitably culturing said yeast or filamentous fungal cell in suitable culture medium, and iii) isolating said secreted protein from said culture medium. Methods for isolating proteins from cultures are known by the person of skill.
  • Culturing methods for producing proteins in yeast or filamentous fungal cells are known by the person of skill, and can be readily adjusted to the present invention. Culturing can be continuous or in batches or fed-batches. Preferred is the method according to the present invention, further comprising suitably inducing the increased expression and/or overexpression or reduced expression and/or inactivation of the at least one fungal gene. Induction can be achieved based on the promotor(s) as used, e.g. by adding inducers, or switching conditions, e.g. temperature.
  • There are many examples of engineering of S. cerevisiae for improved protein production, including optimizing of fermentation process, selecting the expression vectors systems, choosing the signal sequence for extracellular targeting and engineering host strains for better folding and post-translational modification (Tohda H., Kumagai H., Takegawa, K, (2010) Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol 86: 403-417).
  • Preferred is the method according to the present invention, wherein about 30% or more or 40% or more, preferably about 50% or more, more preferably to about 75% or more of said at least one secreted protein is produced, when compared to the production of a control yeast or filamentous fungal cell, preferably one that does not contain a gene as mentioned above leading to increased secretion of the protein of interest
  • Another important aspect of the present invention relates to a method for producing a yeast or filamentous fungal cell producing at least one secreted protein of interest, comprising introducing into said cell producing at least one secreted protein of interest at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, VHS2, ASA1, TRP4, YPS7, CUT824, YOR318C, PRM7, ERV46, FIT2, GPM3, CUT892, SRN2, SUT643, CUT461, THR4, GMH1, SOL1, NAB6, YPR148C, ALP1, CUT097, ATG33, YOR316C-A, SOG2, MCM6, SUT230, SUT419, TIF11, TAF5, PHO91, AIM32, ENO2, UBA2, PUS5, ERG1, SUT311, KSS1, MRP10, CUT598, CUT188, YOR238W, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1 RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, WBP1, AVT2, CUT854, TRM10, SLX9, YPL077C, PET122, TFG2, PUN1, CUT152, AIR2, CUT571, RPS26B, RRT6, RPC19, URA3, YGR045C, SMC3, PNG1, THI6, MEU1, CUT239, NSE4, SUT074, AAH1, RMD5, CUT607, ACS1, MNN1, ARH1, YHR140W, CET1, RRB1, YLR342W-A, RPS22B, CHS5, YIL165C, SUT093, LPX1, NCA3, EFG1, NBP35, CUT765, MSL1, SCD6, ATG42, CHS6, COQ2, RPO31, MKK1, HED1, PBP2, BET5, CUT678, YGR021W, SUT474, YGL159W, IRC21, VHR1, SPP1, PRP43, ZRT1, YLR041W, SUT711, COX18, CBP6, SUT575, CLG1, CUT213, QCR10, SNR3, MSS2, CUT505, YOS1, SUT073, UTP21, ACA1, CUT632, RIP1, HUL5, CUT727, RPL35B, CUT184, CUT420, YFL041W-A, SUT460, ATG10, MFA1, UGX2, TRK2, CUT704, SUT083, TRE1, RVS161, LEA1, EBP2, THI80, CTI6, CUT322, XPT1, MRPL35, YPL025C, SUT737, PGA2, ULP2, MRX16, EST1, NUP100, IES3, ATG39, YMR084W, SUT428, YPL119C-A, MIN8, CUT490, SUT287, KEL3, SUT678, SEC3, SOL4, SIS2, CUT915, RRP3, ESA1, PCL8, TRX3, YKL115C, EMP65, ZDS1, CUT167, SOD1, UBR2, LSP1, SNR81, RGD3, YTP1, SMY2, CUT449, FIN1, YKL106C-A, YAR019W-A, CCH1, AYR1, SUT573, VNX1, FOL3, SUT511, GIS4, CUT743, RPL24A, HMT1, SUT333, SPP2, SUT128, SMC6, PHR1, RPS15, CUT642, GYP7, tK(CUU)K, CUT896, SLM5, CUT586, CUT158, and RRP12, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, YDR262W, CMC1, MRP17, YPT52, CUT312, MRPS5, RDR1, DAL7, RPL20A, YBR137W, RPL36B, YEL008C-A, RAX1, INP51, CUT729, UBP8, CUT258, YLR342W-A, SUT568, PEX7, MSD1, CUT136, TIM10, CUT361, snR51, TAL1, RIP1, MRP10, SUT078, MRP51, GLO3, EHD3, HER1, NMA111, PBP4, MFB1, IKI3, NDL1, SUT433, YOR238W, SUT750, QDR2, RDI1, SUT014, CUT437, MSC6, SUT497, YCR051W, MRPL33, RPL14A, TRM7, RNH202, RTC5, SUT027, CDC5, SUT729, YOR131C, CUT665, GLG2, SUT268, SUT705, MED4, RCR2, EFB1, RXT2, KGD1, TUP1, RNH203, YDR338C, SED1, CUT522, HIS2, SUT145, MET17, APC4, NKP2, MKK2, NDC1, PET100, NIP7, VHT1, SUT685, BNI5, SNA3, EGH1, MRP4, POB3, PIB2, SUT317, and NTO1, wherein said at least one fungal gene shows reduced expression and/or inactivation. Preferably, said at least one fungal gene is integrated into the genome as an expression cassette and/or extrachromosomally expressed, preferably using a replicative expression vector.
  • Preferred is a method of the present invention for producing a yeast or filamentous fungal cell producing at least one secreted protein of interest according to the present invention, comprising introducing into said cell producing at least one secreted protein of interest at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1, RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, and WBP1, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, NDC1, PET100, NIP7, VHT1, and SUT685, wherein said at least one fungal gene shows reduced expression and/or inactivation.
  • Furthermore, the method according to the invention may include further introducing into said cell a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions.
  • In a preferred embodiment according to the method according to the present invention, said method further comprises introducing into said cell the fungal gene PDI1, showing an increased expression and/or overexpression. Preferably, said at least one fungal gene is also integrated into the genome as an expression cassette and/or extrachromosomally expressed, preferably using a replicative expression vector
  • Finally, another important aspect of the present invention relates to the use of a yeast or filamentous fungal cell according to the present invention for producing at least one secreted protein of interest, preferably using a method according to the present invention.
  • In summary, the present invention provides the following items.
  • Item 1. A yeast or filamentous fungal cell producing at least one secreted protein of interest, wherein said cell comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, VHS2, ASA1, TRP4, YPS7, CUT824, YOR318C, PRM7, ERV46, FIT2, GPM3, CUT892, SRN2, SUT643, CUT461, THR4, GMH1, SOL1, NAB6, YPR148C, ALP1, CUT097, ATG33, YOR316C-A, SOG2, MCM6, SUT230, SUT419, TIF11, TAF5, PHO91, AIM32, ENO2, UBA2, PUS5, ERG1, SUT311, KSS1, MRP10, CUT598, CUT188, YOR238W, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1 RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, WBP1, AVT2, CUT854, TRM10, SLX9, YPL077C, PET122, TFG2, PUN1, CUT152, AIR2, CUT571, RPS26B, RRT6, RPC19, URA3, YGR045C, SMC3, PNG1, THI6, MEU1, CUT239, NSE4, SUT074, AAH1, RMD5, CUT607, ACS1, MNN1, ARH1, YHR140W, CET1, RRB1, YLR342W-A, RPS22B, CHS5, YIL165C, SUT093, LPX1, NCA3, EFG1, NBP35, CUT765, MSL1, SCD6, ATG42, CHS6, COQ2, RPO31, MKK1, HED1, PBP2, BET5, CUT678, YGR021W, SUT474, YGL159W, IRC21, VHR1, SPP1, PRP43, ZRT1, YLR041W, SUT711, COX18, CBP6, SUT575, CLG1, CUT213, QCR10, SNR3, MSS2, CUT505, YOS1, SUT073, UTP21, ACA1, CUT632, RIP1, HUL5, CUT727, RPL35B, CUT184, CUT420, YFL041W-A, SUT460, ATG10, MFA1, UGX2, TRK2, CUT704, SUT083, TRE1, RVS161, LEA1, EBP2, THI80, CTI6, CUT322, XPT1, MRPL35, YPL025C, SUT737, PGA2, ULP2, MRX16, EST1, NUP100, IES3, ATG39, YMR084W, SUT428, YPL119C-A, MIN8, CUT490, SUT287, KEL3, SUT678, SEC3, SOL4, SIS2, CUT915, RRP3, ESA1, PCL8, TRX3, YKL115C, EMP65, ZDS1, CUT167, SOD1, UBR2, LSP1, SNR81, RGD3, YTP1, SMY2, CUT449, FIN1, YKL106C-A, YAR019W-A, CCH1, AYR1, SUT573, VNX1, FOL3, SUT511, GIS4, CUT743, RPL24A, HMT1, SUT333, SPP2, SUT128, SMC6, PHR1, RPS15, CUT642, GYP7, tK(CUU)K, CUT896, SLM5, CUT586, CUT158, and RRP12, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, YDR262W, CMC1, MRP17, YPT52, CUT312, MRPS5, RDR1, DAL7, RPL20A, YBR137W, RPL36B, YEL008C-A, RAX1, INP51, CUT729, UBP8, CUT258, YLR342W-A, SUT568, PEX7, MSD1, CUT136, TIM10, CUT361, snR51, TAL1, RIP1, MRP10, SUT078, MRP51, GLO3, EHD3, HER1, NMA111, PBP4, MFB1, IKI3, NDL1, SUT433, YOR238W, SUT750, QDR2, RDI1, SUT014, CUT437, MSC6, SUT497, YCR051W, MRPL33, RPL14A, TRM7, RNH202, RTC5, SUT027, CDC5, SUT729, YOR131C, CUT665, GLG2, SUT268, SUT705, MED4, RCR2, EFB1, RXT2, KGD1, TUP1, RNH203, YDR338C, SED1, CUT522, HIS2, SUT145, MET17, APC4, NKP2, MKK2, NDC1, PET100, NIP7, VHT1, SUT685, BNI5, SNA3, EGH1, MRP4, POB3, PIB2, SUT317, and NTO1, wherein said at least one fungal gene shows reduced expression and/or inactivation.
  • Item 2. The yeast or filamentous fungal cell according to Item 1, wherein said cell comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1, RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, and WBP1, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, NDC1, PET100, NIP7, VHT1, and SUT685, wherein said at least one fungal gene shows reduced expression and/or inactivation.
  • Item 3. The yeast or filamentous fungal cell according to Item 1 or 2, wherein said genes or SUTs or CUTs are furthermore selected from the group of genes or SUTs or CUTs having a value of logFC/FDR of more than 200, more preferred of more than 300, and most preferred of more than 500, based on the values as determined herein.
  • Item 4. The yeast or filamentous fungal cell according to any one of Items 1 to 3, further comprising a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions.
  • Item 5. The yeast or filamentous fungal cell according to any one of Items 1 to 4, further comprising the fungal gene PDI1, showing an increased expression and/or overexpression.
  • Item 6. The yeast or filamentous fungal cell according to any one of Items 1 to 5, wherein said yeast or filamentous fungal cell is selected from the group consisting of Aspergillus spp., Trichoderma spp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces ssp., Pichia spp., Hansenula polymorpha, Fusarium spp., Neurospora spp., and Penicillium spp., preferably Saccharomyces cerevisiae.
  • Item 7. The yeast or filamentous fungal cell according to any one of Items 1 to 6, wherein said at least one secreted protein of interest also shows an increased expression and/or overexpression.
  • Item 8. The yeast or filamentous fungal cell according to any one of Items 1 to 7, wherein said at least one fungal gene showing increased expression and/or overexpression and/or showing reduced expression and/or inactivation is a native gene and/or is a recombinant gene, wherein preferably said recombinant gene is integrated into the genome as an expression cassette and/or extrachromosomally expressed, preferably using a replicative expression vector.
  • Item 9. The yeast or filamentous fungal cell according to any one of Items 1 to 8, wherein the cell furthermore comprises at least one additional recombinant secretion promoting gene, for example a gene for a chaperone, for a foldase and/or for a glycosylation-promoting protein.
  • Item 10. The yeast or filamentous fungal cell according to any one of Items 1 to 9, wherein the increased expression and/or overexpression or reduced expression and/or inactivation of the at least one fungal gene or the at least one additional recombinant secretion promoting gene is constitutive or inducible.
  • Item 11. The yeast or filamentous fungal cell according to any one of Items 1 to 10, wherein the cell produces the at least one secreted protein to about 30% or more, or to about 40% or more, preferably about 50% or more, more preferably to about 75% or more, when compared to a control yeast or filamentous fungal cell.
  • Item 12. A method for producing a secreted protein in a yeast or filamentous fungal cell, comprising the steps of i) providing a yeast or filamentous fungal cell producing at least one secreted protein of interest according to any one of Items 1 to 11, ii) culturing said yeast or filamentous fungal cell in suitable culture medium, and iii) isolating said secreted protein from said culture medium.
  • Item 13. The method according to Item 12, further comprising suitably inducing the increased expression and/or overexpression or reduced expression and/or inactivation of the at least one fungal gene.
  • Item 14. The method according to Item 11 or 12, wherein about 30% or more, or about 40% or more, preferably about 50% or more, more preferably to about 75% or more of said at least one secreted protein is produced, when compared to the production of a control yeast or filamentous fungal cell.
  • Item 15. A method for producing a yeast or filamentous fungal cell producing at least one secreted protein of interest, comprising introducing into said cell producing at least one secreted protein of interest at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1, RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, and WBP1, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, NDC1, PET100, NIP7, VHT1, and SUT685, wherein said at least one fungal gene shows reduced expression and/or inactivation.
  • Item 16. The method according to Item 15, further introducing into said cell a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions.
  • Item 17. The method according to Item 15 or 16, further introducing into said cell the fungal gene PDI1, showing an increased expression and/or overexpression.
  • Item 18. The method according to any one of Items 15 to 17, wherein said at least one fungal gene is integrated into the genome as an expression cassette and/or extrachromosomally expressed, preferably using a replicative expression vector.
  • Item 19. Use of a yeast or filamentous fungal cell according to any one of Items 1 to 10 for producing at least one secreted protein of interest.
  • The present invention will now be described further in the following examples with reference to the accompanying Figure, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.
  • Figure 1 shows the map of plasmid pLI410-062 as used in the methods according to the present invention.
  • Figure 2 A and B shows the results of the α-amylase secretion measurements relative to baseline for selected genes of the present invention as box plots in % control over time (4, 24, 48, and 120 hours). Genes are ALP1, BNA7, GMH1, SUT074, TFG2, ENO2, NMA2, PRY2, and TOM22. HAC1 is control.
  • Figure 3 A and B shows the results of the α-amylase secretion measurements per cell for selected genes of the present invention as box plots in % control over time (4, 24, 48, and 120 hours). Genes are ALP1, BNA7, GMH1, SUT074, TFG2, ENO2, NMA2, PRY2, and TOM22. HAC1 is control.
  • Figure 4 A and B shows the results of the α-amylase secretion measurements (total amylase) for selected genes of the present invention as box plots in % control over time (4, 24, and 48 hours). Genes are INP51, MNT2, TLG2, TPO2, and YDR262W. HAC1, HDA2 and ER.sec2 are controls.
  • Figure 5 A and B shows the results of the α-amylase secretion measurements per cell for selected genes of the present invention as box plots in % control overtime (4, 24, and 48 hours). Genes are INP51, MNT2, TLG2, TPO2, and YDR262W. HAC1, HDA2 and ER.sec2 are controls.
    • Examples Materials and Methods Construction of yeast overexpression strains
  • For overexpression of target genes using genome integration, candidate genes were cloned into plasmid pLI410-062 between the AscI and SbfI restriction sites, which was then linearized by NotI enzyme, and transformed into yeast strain ER.sec2. The plasmid integrates into the yeast chromosome at the BUD5 locus (Figure 1). For plasmid based overexpression of target genes, native candidate genes were cloned into plasmid p427-TEF between SpeI and SalI and transformed into yeast strain ER.sec2.
    • Construction of yeast deletion strains
  • Deletion strains were constructed by golden gate assembly of annealed oligos with gRNA sequences targeting the start and end position of the target gene, into sgRNA expression vector pWS082. The assembled plasmid and Cas9 expression vector pWS173 were linearized using EcoRV or BsmBI and co-transformed with annealed repair fragments, consisting of the joined 60 bp flanking regions of each target gene, which upon successful homology directed repair, resulted in the deletion of the target gene in ER.sec2.
    • α-Amylase activity measurement
  • Preculture of YPD was performed, either with 22h of culture on SD-2xSCAA, or 22h and 96 h of culture on YPD. SD-2×SCAA medium was prepared as described previously (Hackel et al. 2006; Tyo et al. 2012), and the composition of SD-2xSCAA was as follows: 10 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids, 2 g/L, KH2PO4 (pH 6.0 by NaOH), and 1 g/L BSA, containing filter sterilized SCAA solution (190 mg/L arginine, 108 mg/L methionine, 52 mg/L tyrosine, 290 mg/L isoleucine, 440 mg/L lysine, 200 mg/L phenylalanine, 1,260 mg/L, glutamic acid, 400 mg/L aspartic acid, 380 mg/L valine, 220 mg/L threonine, 130 mg/L glycine, 400 mg/L leucine, 40 mg/L tryptophan, and 140 mg/L histidine) (see Liu et al., 2013 - Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae).
  • The initial OD600nm was 0.1, and flasks of 250 ml + 50 ml of medium were used. Culture density was measured at OD600nm.
  • For the assay, 100 µL of supernatant + 900 µL of acetate buffer 50mM pH5.5 were combined, and 10 µL of sample were incubated for 5 min at 40°C in a PCR well plate.
  • Afterwards, 10 µL of BPNPG7 substrate was added, followed by incubation for 10 min at 40°C. The reaction was stopped by adding 150 µL of Trizma base 1%, followed by vortexing. The result was read at an OD of 400nm, which generally required a prior step of 10 or 20-fold dilution.
    • Calculation of α-amylase activity
  • The activity U was calculated as U = (ΔE400/10) × (0.17/0.01) × (1/18.1) × D
  • E400 : Sample absorbance - blank absorbance, 10 : time of reaction, 0.17 : total volume of reaction, 0.01 : volume of sample, 18.1 : EmM p-nitrophenol in Trizma base 1%, D : Dilution of sample. Normalization of α-amylase activity was performed with respective OD600nm.
    • Results
  • The following genes and SUTs (stable uncharacterized transcripts) or CUTs (cryptic unstable transcripts) were identified as being of relevance, and relevance was defined as at least 2% increase of amylase activity (see above). See also Figure 2.
  • 1. Genes that were activated/overexpressed (integration of overexpression cassette into the genome and/or overexpression through a replicative plasmid) after statistical and enrichment analysis - preferred selection. logFC (log fold change) indicates the measure of enrichment, a higher value, equals a higher enrichment in the experiments as performed. FDR (false discovery rate) indicates the corrected p-value, a lower value means less variance between replicates as performed.
    Gene (common name, SUT or CUT or systematic designation) Name and function (if known) logFC FDR
    MIC19 Component of the MICOS complex 13.883 0.036
    TOM22 Translocase of the Outer Mitochondrial membrane; responsible for initial import of mitochondrially directed proteins 13.781 0.008
    NKP1 Non-essential Kinetochore Protein 13.389 0.012
    DML1 Drosophila melanogaster Misato-Like protein, Essential protein involved in mtDNA inheritance 13.307 0.014
    CUT859 SUT or CUT 13.152 0.033
    GAL80 GALactose metabolism, Transcriptional regulator involved in the repression of GAL genes 12.170 0.008
    APM3 clathrin Adaptor Protein complex Medium chain 12.088 0.020
    COQ10 COenzyme Q, Coenzyme Q (ubiquinone) binding protein 12.048 0.025
    BLM10 BLeoMycin resistance, Proteasome activator 12.008 0.030
    MDH1 Malate DeHydrogenase, Mitochondrial malate dehydrogenase 11.915 0.008
    VHS2 Viable in a Hal3 Sit4 background, Regulator of septin dynamics 11.838 0.032
    ASA1 AStra Associated protein, Subunit of the ASTRA complex 11.801 0.015
    TRP4 TRyPtophan, Anthranilate phosphoribosyl transferase 11.698 0.019
    YPS7 YaPSin, Putative GPI-anchored aspartic protease 11.620 0.030
    CUT824 SUT or CUT 11.529 0.041
    YOR318C Gene of unknown function 11.515 0.013
    PRM7 Pheromone-Regulated Membrane protein 11.485 0.023
    ERV46 ER Vesicle, Protein localized to COPII-coated vesicles 11.350 0.010
    FIT2 Facilitator of Iron Transport, Mannoprotein that is incorporated into the cell wall 11.287 0.034
    GPM3 Glycerate PhosphoMutase 11.062 0.019
    CUT892 SUT or CUT 10.972 0.050
    SRN2 Suppressor of Rna mutations, Number 2 10.938 0.021
    SUT643 SUT or CUT 10.910 0.039
    CUT461 SUT or CUT 10.901 0.042
    THR4 THReonine requiring, Threonine synthase 10.840 0.047
    GMH1 Gea1-6 Membrane-associated High-copy suppressor; Golgi membrane protein of unknown function 10.780 0.055
    SOL1 Suppressor Of Los1-1, Protein with a possible role in tRNA export 10.725 0.026
    NAB6 Nucleic Acid Binding protein, Putative RNA-binding protein 10.674 0.013
    YPR148C Gene of unknown function 10.614 0.027
    ALP1 Arginine transporter 10.598 0.046
    CUT097 SUT or CUT 10.597 0.046
    ATG33 AuTophaGy related, Mitochondrial mitophagy-specific protein 10.585 0.030
    YOR316C-A Gene of unknown function 10.547 0.025
    SOG2 Key component of the RAM signaling network; required for proper cell morphogenesis and cell separation after mitosis 10.546 0.039
    MCM6 MiniChromosome Maintenance, Protein involved in DNA replication 10.531 0.019
    SUT230 SUT or CUT 10.507 0.010
    SUT419 SUT or CUT 10.398 0.027
    TIF11 Translation Initiation Factor 10.334 0.024
    TAF5 TATA binding protein-Associated Factor, involved in RNA polymerase II transcription initiation and in chromatin modification 10.328 0.027
    PHO91 PHOsphate metabolism, Low-affinity vacuolar phosphate transporter 10.303 0.024
    AIM32 Altered Inheritance rate of Mitochondria, 2Fe-2S mitochondrial protein involved in redox quality control 10.271 0.042
    ENO2 ENOlase, Enolase II, a phosphopyruvate hydratase 10.260 0.050
    UBA2 UBiquitin Activating, Subunit of heterodimeric nuclear SUMO activating enzyme E1 with Aos1p 10.215 0.030
    PUS5 PseudoUridine Synthase 10.197 0.030
    ERG1 ERGosterol biosynthesis, Squalene epoxidase 10.139 0.013
    SUT311 SUT or CUT 10.130 0.012
    KSS1 Kinase Suppressor of Sst2 mutations, Mitogen-activated protein kinase (MAPK) 10.116 0.039
    MRP10 Mitochondrial Ribosomal Protein, Mitochondrial ribosomal protein of the small subunit 10.099 0.023
    CUT598 SUT or CUT 10.099 0.046
    CUT188 SUT or CUT 10.073 0.026
    YOR238W Gene of unknown function 10.023 0.025
    EMW1 Essential for Maintenance of the cell Wall, Essential conserved protein with a role in cell wall integrity 15.549 0.071
    BNA7 Biosynthesis of NAD, Formylkynurenine formamidase 14.863 0.071
    SNR63 Small Nucleolar RNA, C/D box small nucleolar RNA (snoRNA) 14.717 0.071
    CCT3 Chaperonin Containing TCP-1, Subunit of the cytosolic chaperonin Cct ring complex 14.647 0.071
    PRY2 Pathogen Related in Yeast, Sterol binding protein involved in the export of acetylated sterols 14.548 0.071
    MAL11 MALtose fermentation, High-affinity maltose transporter (alpha-glucoside transporter) 14.484 0.071
    KRS1 Lysyl (K) tRNA Synthetase 14.290 0.072
    RAI1 Rat1p Interacting Protein, Nuclear decapping endonuclease 14.254 0.071
    SUT784 SUT or CUT 13.682 0.071
    YPR148C Gene of unknown function 13.572 0.071
    YEL1 Yeast EFA6-Like, Guanine nucleotide exchange factor specific for Arf3p 13.417 0.096
    CUT832 SUT or CUT 13.118 0.071
    NMA2 Nicotinamide Mononucleotide Adenylyltransferase 13.116 0.071
    VPS27 Vacuolar Protein Sorting, Endosomal protein that forms a complex with Hse1p 12.963 0.071
    SUT428 SUT or CUT 12.841 0.089
    PEX29 PEroXisome related, ER-resident protein involved in peroxisomal biogenesis 12.477 0.071
    YLR446W Gene of unknown function 12.369 0.071
    WBP1 Wheat germ agglutinin-Binding Protein, Beta subunit of the oligosaccharyl transferase glycoprotein complex 12.078 0.087
    AVT2 Amino acid Vacuolar Transport, Putative transporter 10.965 0.071
    CUT854 SUT or CUT 10.873 0.093
    TRM10 Transfer RNA Methyltransferase, methylates the N-1 position of guanine at position 9 in tRNAs 10.442 0.099
    SLX9 Protein required for pre-rRNA processing 9.996 0.012
    YPL077C Gene of unknown function 9.994 0.038
    PET122 PETite colonies, Mitochondrial translational activator specific for the COX3 mRNA 9.982 0.039
    TFG2 Transcription Factor G; involved in both transcription initiation and elongation of RNA polymerase II 9.973 0.090
    PUN1 Plasma membrane protein Upregulated during Nitrogen stress 9.950 0.027
    CUT152 SUT or CUT 9.936 0.020
    AIR2 Arginine methyltransferase-Interacting RING finger protein, involved in nuclear RNA processing and degradation 9.886 0.044
    CUT571 SUT or CUT 9.799 0.033
    RPS26B Protein component of the small (40S) ribosomal subunit 9.789 0.023
    RRT6 Regulator of rDNA Transcription 9.749 0.012
    RPC19 RNA Polymerase C, RNA polymerase subunit AC19 9.715 0.047
    URA3 URAcil requiring, Orotidine-5'-phosphate (OMP) decarboxylase 9.687 0.046
    YGR045C Gene of unknown function 9.679 0.039
    SMC3 Stability of MiniChromosomes, Subunit of the multiprotein cohesin complex 9.669 0.025
    PNG1 Peptide N-Glycanase 9.654 0.019
    THI6 THIamine biosynthesis, Thiamine-phosphate diphosphorylase and hydroxyethylthiazole kinase 9.653 0.033
    MEU1 Multicopy Enhancer of UAS2, Methylthioadenosine phosphorylase (MTAP) 9.558 0.031
    CUT239 SUT or CUT 9.531 0.032
    NSE4 Non-SMC Element, Component of the SMC5-SMC6 complex 9.502 0.023
    SUT074 SUT or CUT 9.478 0.019
    AAH1 Adenine AminoHydrolase, Adenine deaminase (adenine aminohydrolase) 9.454 0.044
    RMD5 Required for Meiotic nuclear Division, Component of GID Complex that confers ubiquitin ligase (U3) activity 9.452 0.024
    CUT607 SUT or CUT 9.313 0.020
    ACS1 Acetyl CoA Synthetase, Acetyl-coA synthetase isoform 9.305 0.036
    MNN1 MaNNosyltransferase, Alpha-1,3-mannosyltransferase 9.265 0.019
    ARH1 Adrenodoxin Reductase Homolog, Oxidoreductase of the mitochondrial inner membrane 9.244 0.039
    YHR140W Gene of unknown function 9.220 0.021
    CET1 Capping Enzyme Triphosphatase, RNA 5'-triphosphatase involved in mRNA 5' capping 9.203 0.019
    RRB1 Regulator of Ribosome Biogenesis, Specific assembly chaperone for ribosomal protein Rpl3p 9.185 0.030
    YLR342W-A Gene of unknown function 9.166 0.010
    RPS22B Ribosomal Protein of the Small subunit, Protein component of the small (40S) ribosomal subunit 9.154 0.024
    CHS5 CHitin Synthase-related, Component of the exomer complex 9.143 0.027
    YIL165C Gene of unknown function 9.140 0.040
    SUT093 SUT or CUT 9.139 0.030
    LPX1 Lipase of PeroXisomes, Peroxisomal matrix-localized lipase 9.114 0.039
    NCA3 Nuclear Control of ATPase, Protein involved in mitochondrion organization 9.078 0.026
    EFG1 Exit From G1, Ribosome biogenesis factor required for maturation of 18S rRNA 9.063 0.040
    NBP35 Nucleotide Binding Protein, Essential cytoplasmic iron-sulfur cluster binding protein 9.055 0.042
    CUT765 SUT or CUT 9.038 0.037
    MSL1 MUD Synthetic Lethal 9.019 0.015
    SCD6 Suppressor of Clathrin Deficiency, Repressor of translation initiation 9.004 0.025
    ATG42 AuTophaGy, Vacuolar serine-type carboxypeptidase 9.001 0.028
    CHS6 CHitin Synthase-related, Member of the ChAPs (Chs5p-Arflp-binding proteins) family 8.974 0.020
    COQ2 COenzyme Q, Para hydroxybenzoate polyprenyl transferase 8.973 0.045
    RPO31 RNA Polymerase, RNA polymerase III largest subunit C160 8.969 0.044
    MKK1 Mitogen-activated protein Kinase-Kinase, MAPKK involved in the protein kinase C signaling pathway 8.958 0.030
    HED1 High copy suppressor of rED1, Meiosis-specific protein 8.903 0.025
    PBP2 Pbp1p Binding Protein, RNA binding protein; has similarity to mammalian heterogeneous nuclear RNP K protein 8.891 0.027
    BET5 Blocked Early in Transport, Core component of transport protein particle (TRAPP) complexes I-III 8.890 0.019
    CUT678 SUT or CUT 8.876 0.045
    YGR021W Gene of unknown function 8.823 0.012
    SUT474 SUT or CUT 8.811 0.042
    YGL159W Gene of unknown function 8.802 0.014
    IRC21 Increased Recombination Centers, unknown function 8.795 0.027
    VHR1 VHt1 Regulator, Transcriptional activator 8.760 0.046
    SPP1 Setlc, Phd finger Protein, Subunit of COMPASS (Set1C) 8.721 0.025
    PRP43 Pre-mRNA Processing, RNA helicase in the DEAH-box family 8.707 0.042
    ZRT1 Zinc-Regulated Transporter, High-affinity zinc transporter of the plasma membrane; responsible for the majority of zinc uptake 8.705 0.039
    YLR041W Gene of unknown function 8.687 0.044
    SUT711 SUT or CUT 8.686 0.039
    COX18 Cytochrome c OXidase, Protein required for membrane insertion of C-terminus of Cox2p 8.685 0.046
    CBP6 Cytochrome B Protein synthesis, Mitochondrial protein required for translation of the COB mRNA 8.678 0.043
    SUT575 SUT or CUT 8.651 0.042
    CLG1 Cyclin-Like Gene, Cyclin-like protein that interacts with Pho85p 8.651 0.047
    CUT213 SUT or CUT 8.610 0.036
    QCR10 ubiQuinol-cytochrome C oxidoReductase, Subunit of the ubiqunol-cytochrome c oxidoreductase complex 8.604 0.019
    SNR3 Small Nucleolar RNA, H/ACA box small nucleolar RNA (snoRNA) 8.571 0.044
    MSS2 Mitochondrial Splicing, Peripherally bound inner membrane protein of the mitochondrial matrix 8.559 0.023
    CUT505 SUT or CUT 8.557 0.039
    YOS1 Yip One Suppressor, Integral membrane protein required for ER to Golgi transport 8.540 0.023
    SUT073 SUT or CUT 8.519 0.033
    UTP21 U Three Protein, Subunit of U3-containing 90S preribosome and SSU processome complexes 8.511 0.039
    ACA1 ATF/CREB Activator, ATF/CREB family basic leucine zipper (bZIP) transcription factor 8.478 0.045
    CUT632 SUT or CUT 8.475 0.039
    RIP1 Rieske Iron-sulfur Protein, Ubiquinol-cytochrome-c reductase 8.466 0.037
    HUL5 Hect Ubiquitin Ligase, Multiubiquitin chain assembly factor (E4) 8.383 0.042
    CUT727 SUT or CUT 8.373 0.030
    RPL35B Ribosomal 60S subunit protein L35B 8.360 0.019
    CUT 184 SUT or CUT 8.304 0.039
    CUT420 SUT or CUT 8.300 0.023
    YFL041W-A Gene of unknown function 8.290 0.010
    SUT460 SUT or CUT 8.248 0.023
    ATG10 AuTophaGy related, Conserved E2-like conjugating enzyme 8.244 0.019
    MFA1 Mating Factor A, Mating pheromone α-factor 8.231 0.023
    UGX2 Protein of unknown function 8.226 0.023
    TRK2 TRansport of potassium (K), Component of the Trklp-Trk2p potassium transport system 8.218 0.027
    CUT704 SUT or CUT 8.201 0.041
    SUT083 SUT or CUT 8.189 0.025
    TRE1 Transferrin REceptor like, Transferrin receptor-like protein 8.183 0.046
    RVS161 Reduced Viability on Starvation, Amphiphysin-like lipid raft protein 8.110 0.034
    LEA1 Looks Exceptionally like U2A, Component of U2 snRNP complex 8.092 0.044
    EBP2 EBNA1-binding protein (homolog), Required for 25S rRNA maturation and 60S ribosomal subunit assembly; 8.089 0.030
    THI80 THIamine metabolism, Thiamine pyrophosphokinase 8.071 0.012
    CTI6 Cyc8-Tup1 Interacting protein, Component of the Rpd3L histone deacetylase complex 8.065 0.019
    CUT322 SUT or CUT 8.002 0.027
    XPT1 Xanthine Phosphoribosyl Transferase, Xanthine-guanine phosphoribosyl transferase 7.984 0.036
    MRPL35 Mitochondrial Ribosomal Protein, Large subunit 7.963 0.031
    YPL025C Gene of unknown function 7.962 0.037
    SUT737 SUT or CUT 7.950 0.025
    PGA2 Processing of Gas1p and ALP, Essential protein required for maturation of Gas1p and Pho8p 7.941 0.046
    ULP2 UbL-specific Protease, Peptidase that deconjugates Smt3/SUMO-1 peptides from proteins 7.935 0.033
    MRX16 Mitochondrial oRganization of gene eXpression (MIOREX), Protein that associates with the large mitoribosomal subunit 7.917 0.044
    EST1 Ever Shorter Telomeres, TLC1 RNA-associated factor involved in telomere length regulation 7.911 0.042
    NUP100 NUclear Pore, FG-nucleoporin component of central core of the nuclear pore complex 7.902 0.021
    IES3 Ino Eighty Subunit, Subunit of the INO80 chromatin remodeling complex 7.880 0.031
    ATG39 AuTophaGy related, Autophagy receptor with a role in degradation of the ER and nucleus 7.876 0.038
    YMR084W Gene of unknown function 7.850 0.027
    SUT428 SUT or CUT 7.827 0.030
    YPL119C-A Gene of unknown function 7.791 0.031
    MIN8 mitochondrial MINi protein of 8 kDa 7.783 0.027
    CUT490 SUT or CUT 7.779 0.045
    SUT287 SUT or CUT 7.708 0.027
    KEL3 KELch 7.705 0.027
    SUT678 SUT or CUT 7.699 0.025
    SEC3 SECretory, Subunit of the exocyst complex 7.691 0.045
    SOL4 Suppressor Of Los1-1, 6-phosphogluconolactonase 7.678 0.030
    SIS2 SIt4 Suppressor, Negative regulatory subunit of protein phosphatase 1 (Ppz1p) 7.650 0.026
    CUT915 SUT or CUT 7.649 0.044
    RRP3 Ribosomal RNA Processing, Protein involved in rRNA processing 7.635 0.034
    ESA1 Catalytic subunit of the histone acetyltransferase complex (NuA4) 7.612 0.031
    PCL8 Pho85 CycLin, Cyclin 7.581 0.046
    TRX3 ThioRedoXin, Mitochondrial thioredoxin 7.579 0.033
    YKL115C Gene of unknown function 7.530 0.043
    EMP65 ER Membrane Protein of 65 kDa, Integral membrane protein of the ER 7.520 0.029
    ZDS1 Zillion Different Screens, Protein with a role in regulating Swe1p-dependent polarized growth 7.488 0.049
    CUT167 SUT or CUT 7.486 0.016
    SOD1 SuperOxide Dismutase, Cytosolic copper-zinc superoxide dismutase 7.471 0.019
    UBR2 Cytoplasmic ubiquitin-protein ligase (E3 7.470 0.044
    LSP1 Long chain bases Stimulate Phosphorylation, Eisosome core component 7.391 0.031
    SNR81 H/ACA box small nucleolar RNA (snoRNA) 7.389 0.030
    RGD3 GTPase activating protein (GAP) for Rho3p 7.370 0.032
    YTP1 Yeast putative Transmembrane Protein, Probable type-III integral membrane protein of unknown function 7.365 0.042
    SMY2 Suppressor of MYo2-66, involved in COPII vesicle formation 7.352 0.034
    CUT449 SUT or CUT 7.340 0.024
    FIN1 Filaments In between Nuclei, Spindle pole body-related intermediate filament protein 7.335 0.039
    YKL106C-A Gene of unknown function 7.293 0.021
    YAR019W-A Gene of unknown function 7.280 0.019
    CCH1 Calcium Channel Homolog, Voltage-gated high-affinity calcium channel 7.270 0.031
    AYR1 1-AcyldihYdroxyacetone-phosphate Reductase, ifunctional triacylglycerol lipase and 1-acyl DHAP reductase 7.243 0.012
    SUT573 SUT or CUT 7.234 0.042
    VNX1 Vacuolar Na+/H+ eXchanger, Calcium/H+ antiporter localized to the endoplasmic reticulum membrane 7.232 0.010
    FOL3 FOLic acid synthesis, Dihydrofolate synthetase 7.215 0.032
    SUT511 SUT or CUT 7.212 0.026
    GIS4 GIg1-2 Suppressor, proposed to be involved in the RAS/cAMP signaling pathway 7.196 0.027
    CUT743 SUT or CUT 7.171 0.034
    RPL24A Ribosomal 60S subunit protein L24A 7.169 0.039
    HMT1 HnRNP MethylTransferase, Nuclear SAM-dependent mono- and asymmetric methyltransferase 7.163 0.026
    SUT333 SUT or CUT 7.141 0.031
    SPP2 Suppressor of PrP, Essential protein that promotes the first step of splicing 7.137 0.027
    SUT128 SUT or CUT 7.120 0.049
    SMC6 Structural Maintenance of Chromosomes, Subunit of the SMC5-SMC6 complex 7.120 0.047
    PHR1 PHotoreactivation Repair deficient, DNA photolyase involved in photoreactivation 7.119 0.030
    RPS15 Protein component of the small (40S) ribosomal subunit 7.072 0.012
    CUT642 SUT or CUT 7.066 0.025
    GYP7 Gtpase-activating protein for Ypt7 Protein, GTPase-activating protein for yeast Rab family members 7.063 0.021
    tK(CUU)K Lysine tRNA (tRNA-Lys) 7.034 0.041
    CUT896 SUT or CUT 7.026 0.041
    SLM5 Synthetic Lethal with Mss4, Mitochondrial asparaginyl-tRNA synthetase 7.024 0.039
    CUT586 SUT or CUT 7.020 0.038
    CUT158 SUT or CUT 7.003 0.030
    RRP12 Ribosomal RNA Processing, Protein required for export of the ribosomal subunits 7.002 0.031
  • 1a. Gene to be preferably combined with the preferred selection
    PDI1 Protein Disulfide Isomerase 12.524 0.072
  • 2. Genes or SUTs or CUTs that were inactivated/repressed after statistical and enrichment analysis - preferred selection. logFC (log fold change) indicates the measure of enrichment, a higher value, equals a higher enrichment in the experiments as performed. FDR (false discovery rate) indicates the corrected p-value, a lower value means less variance between replicates as performed.
    Gene (common name, SUT or CUT or systematic designation) Name and function (if known) logFC FDR
    TLG2 T-snare affecting a Late Golgi compartment, Syntaxin-like t-SNARE 13.51 0.010
    CUT901 SUT or CUT 11.72 0.009
    ATG33 AuTophaGy related, Mitochondrial mitophagy-specific protein 11.53 0.009
    THR4 THReonine requiring, Threonine synthase 11.49 0.009
    YDR262W Gene of unknown function 10.92 0.009
    CMC1 Cx9C Mitochondrial protein necessary for full assembly of Cytochrome c oxidase, Copper-binding protein of the mitochondrial intermembrane space 10.86 0.009
    MRP17 Mitochondrial ribosomal protein of the small subunit 10.20 0.019
    YPT52 Yeast Protein Two, Endosomal Rab family GTPase; required for vacuolar protein sorting 8.91 0.043
    CUT312 SUT or CUT 8.90 0.014
    MRPS5 Mitochondrial Ribosomal Protein, Small subunit 8.87 0.022
    RDR1 Repressor of Drug Resistance, Transcriptional repressor involved in regulating multidrug resistance 8.65 0.042
    DAL7 Degradation of Allantoin, Malate synthase 8.55 0.009
    RPL20A Ribosomal 60S subunit protein L20A 8.19 0.025
    YBR137W Gene of unknown function 8.11 0.056
    RPL36B Ribosomal 60S subunit protein L36B 8.02 0.028
    YEL008C-A Gene of unknown function 7.85 0.062
    RAX1 Revert to Axial, Protein involved in establishing bud site selection 7.65 0.019
    INP51 INositol polyphosphate 5-Phosphatase 7.51 0.102
    CUT729 SUT or CUT 7.27 0.066
    UBP8 UBiquitin-specific processing Protease, Ubiquitin-specific protease component of the SAGA acetylation complex 7.18 0.066
    CUT258 SUT or CUT 7.10 0.089
    YLR342W-A Gene of unknown function 7.09 0.025
    SUT568 SUT or CUT 7.04 0.027
    PEX7 PEroXin, Peroxisomal signal receptor for peroxisomal matrix proteins 7.00 0.024
    MSD1 Mitochondrial aminoacyl-tRNA Synthetase, Aspartate (D) 6.97 0.089
    CUT136 SUT or CUT 6.88 0.039
    TIM10 Translocase of the Inner Membrane, Essential protein of the mitochondrial intermembrane space 6.84 0.064
    CUT361 SUT or CUT 6.83 0.037
    snR51 Small Nucleolar RNA 6.80 0.085
    TAL1 TransALdolase, Transaldolase, enzyme in the non-oxidative pentose phosphate pathway 6.74 0.069
    RIP1 Rieske Iron-sulfur Protein, Ubiquinol-cytochrome-c reductase 6.65 0.058
    MRP10 Mitochondrial Ribosomal Protein, Mitochondrial ribosomal protein of the small subunit 6.63 0.051
    SUT078 SUT or CUT 6.52 0.074
    MRP51 Mitochondrial Ribosomal Protein, Mitochondrial ribosomal protein of the small subunit 6.51 0.065
    GLO3 GLyOxalase, ADP-ribosylation factor GTPase activating protein (ARF GAP); involved in ER-Golgi transport 6.51 0.053
    EHD3 3-hydroxyisobutyryl-CoA hydrolase 6.50 0.025
    HER1 Hmg2p ER Remodeling, Protein of unknown function 6.48 0.051
    NMA111 Nuclear Mediator of Apoptosis, Serine protease and general molecular chaperone 6.45 0.041
    PBP4 Pbp1p binding protein 6.28 0.044
    MFB1 Mitochondria-associated F-box protein; involved in maintenance of normal mitochondrial morphology 6.25 0.098
    IKI3 Insensitive to KIller toxin, Subunit of Elongator complex 6.21 0.031
    NDL1 NuDeL homolog, Homolog of nuclear distribution factor NudE 6.15 0.057
    SUT433 SUT or CUT 5.99 0.022
    YOR238W Gene of unknown function 5.91 0.054
    SUT750 SUT or CUT 5.86 0.016
    QDR2 QuiniDine Resistance, Plasma membrane transporter of the major facilitator superfamily 5.84 0.020
    RDI1 Rho GDP Dissociation Inhibitor 5.79 0.023
    SUT014 SUT or CUT 5.76 0.059
    CUT437 SUT or CUT 5.75 0.045
    MSC6 Meiotic Sister-Chromatid recombination, Multicopy suppressor of HER2 involved in mitochondrial translation 5.66 0.055
    SUT497 SUT or CUT 5.54 0.072
    YCR051W Gene of unknown function 5.52 0.076
    MRPL33 Mitochondrial Ribosomal Protein, Large subunit 5.47 0.024
    RPL14A Ribosomal 60S subunit protein L14A 5.46 0.077
    TRM7 2'-O-ribose methyltransferase 5.43 0.081
    RNH202 Ribonuclease H2 subunit; required for RNase H2 activity 5.43 0.083
    RTC5 Restriction of Telomere Capping, Protein of unknown function 5.38 0.060
    SUT027 SUT or CUT 5.34 0.058
    CDC5 Cell Division Cycle, Polo-like kinase essential for mitotic cell cycle 5.33 0.070
    SUT729 SUT or CUT 5.30 0.076
    YOR131C Gene of unknown function 5.28 0.078
    CUT665 SUT or CUT 5.12 0.097
    GLG2 Glycogenin-Like Gene, Glycogenin glucosyltransferase 5.12 0.079
    SUT268 SUT or CUT 4.89 0.087
    SUT705 SUT or CUT 4.87 0.086
    MED4 MEDiator complex, Subunit of the RNA polymerase II mediator complex 4.61 0.093
    RCR2 Resistance to Congo Red, Vacuolar ubiquitin ligase-substrate adaptor 4.59 0.055
    EFB1 Elongation Factor Beta, Translation elongation factor 1 beta 4.58 0.036
    RXT2 Component of the histone deacetylase Rpd3L complex 4.49 0.073
    KGD1 alpha-KetoGlutarate Dehydrogenase, Subunit of the mitochondrial alpha-ketoglutarate dehydrogenase complex 4.42 0.093
    TUP1 dTMP-UPtake, General repressor of transcription 4.35 0.080
    RNH203 Ribonuclease H2 subunit 4.31 0.096
    YDR338C Gene of unknown function 3.92 0.029
    SED1 Suppression of Exponential Defect, Major stress-induced structural GPI-cell wall glycoprotein 3.81 0.089
    CUT522 SUT or CUT 3.75 0.092
    HIS2 HIStidine requiring, Histidinolphosphatase 3.74 0.090
    SUT145 SUT or CUT 3.67 0.072
    MET17 METhionine requiring, O-acetyl homoserine-O-acetyl serine sulfhydrylase 3.58 0.063
    APC4 Anaphase Promoting, Subunit of the Anaphase-Promoting Complex/Cyclosome (APC/C) 3.58 0.077
    NKP2 Non-essential Kinetochore Protein, Central kinetochore protein and subunit of the Ctf19 complex 3.54 0.022
    MKK2 Mitogen-activated Kinase Kinase, MAPKK involved in the protein kinase C signaling pathway 3.05 0.042
    NDC1 Nuclear Division Cycle, Subunit of the transmembrane ring of the nuclear pore complex (NPC) 14.18 0.079
    PET100 PETite colonies, Chaperone that facilitates the assembly of cytochrome c oxidase 12.69 0.086
    NIP7 Nuclear ImPort, Nucleolar protein required for 60S ribosome subunit biogenesis 12.54 0.086
    VHT1 Vitamin H Transporter, High-affinity plasma membrane H+-biotin (vitamin H) symporter 12.31 0.086
    SUT685 SUT or CUT 12.07 0.086
    BNI5 Bud Neck Involved, Linker protein responsible for recruitment of myosin to the bud neck 11.96 0.086
    SNA3 Sensitivity to NA+, Protein involved in efficient MVB sorting of proteins to the vacuole 11.93 0.086
    EGH1 Cryptococcus neoformans EGCrP2 Homolog, Steryl-beta-glucosidase with broad specificity for aglycones 11.81 0.086
    MRP4 Mitochondrial ribosomal protein of the small subunit 11.67 0.086
    POB3 POl1 Binding, Subunit of the heterodimeric FACT complex (Spt16p-Pob3p) 10.87 0.086
    PIB2 PtdIns(3)p-Binding, Phosphatidylinositol 3-phosphate binding protein 10.80 0.086
    SUT317 SUT or CUT 10.74 0.086
    NTO1 NuA Three Orf, Subunit of the NuA3 histone acetyltransferase complex 10.62 0.086
  • 3. Genes that were either overexpressed or inactivated/repressed depending on experimental conditions after statistical and enrichment analysis - preferred selection. logFC (log fold change) indicates the measure of enrichment, a higher value, equals a higher enrichment in the experiments as performed. FDR (false discovery rate) indicates the corrected p-value, a lower value means less variance between replicates as performed.
    Gene (common name, SUT or CUT or designation) Name and function (if known) logFC activation logFC repression FDR activation FDR repression
    THR4 THReonine requiring, Threonine synthase 10.84 11.49 0.047 0.009
    MRP10 Mitochondrial Ribosomal Protein, Mitochondrial ribosomal protein of the small subunit 10.10 6.63 0.023 0.051
    RIP1 Rieske Iron-sulfur Protein, Ubiquinol-cytochrome-c reductase 8.47 6.65 0.037 0.058
    YLR342W-A Gene of unknown function 9.17 7.09 0.010 0.025
    ATG33 AuTophaGy related, Mitochondrial 10.59 11.53 0.030 0.009
    mitophagy-specific protein
    YOR238W Gene of unknown function 10.02 5.91 0.025 0.054
  • 3. Genes that were overexpressed - particularly preferred selection
    Gene (common name, SUT or CUT or systematic designation) Name and function (if known) logFC FDR
    MIC19 Component of the MICOS complex 13.883 0.036
    TOM22 Translocase of the Outer Mitochondrial membrane; responsible for initial import of mitochondrially directed proteins 13.781 0.008
    NKP1 Non-essential Kinetochore Protein 13.389 0.012
    DML1 Drosophila melanogaster Misato-Like protein, Essential protein involved in mtDNA inheritance 13.307 0.014
    CUT859 SUT or CUT 13.152 0.033
    GAL80 GALactose metabolism, Transcriptional regulator involved in the repression of GAL genes 12.170 0.008
    APM3 clathrin Adaptor Protein complex Medium chain 12.088 0.020
    COQ10 COenzyme Q, Coenzyme Q (ubiquinone) binding protein 12.048 0.025
    BLM10 BLeoMycin resistance, Proteasome activator 12.008 0.030
    MDH1 Malate DeHydrogenase, Mitochondrial malate dehydrogenase 11.915 0.008
    VHS2 Viable in a Hal3 Sit4 background, Regulator of septin dynamics 11.838 0.032
    ASA1 AStra Associated protein, Subunit of the ASTRA complex 11.801 0.015
    TRP4 TRyPtophan, Anthranilate phosphoribosyl transferase 11.698 0.019
    YPS7 YaPSin, Putative GPI-anchored aspartic protease 11.620 0.030
    CUT824 SUT or CUT 11.529 0.041
    YOR318C Gene of unknown function 11.515 0.013
    PRM7 Pheromone-Regulated Membrane protein 11.485 0.023
    ERV46 ER Vesicle, Protein localized to COPII-coated vesicles 11.350 0.010
    FIT2 Facilitator of Iron Transport, Mannoprotein that is incorporated into the cell wall 11.287 0.034
    GPM3 Glycerate PhosphoMutase 11.062 0.019
    CUT892 SUT or CUT 10.972 0.050
    SRN2 Suppressor of Rna mutations, Number 2 10.938 0.021
    SUT643 SUT or CUT 10.910 0.039
    CUT461 SUT or CUT 10.901 0.042
    THR4 THReonine requiring, Threonine synthase 10.840 0.047
    GMH1 Gea1-6 Membrane-associated High-copy suppressor; Golgi membrane protein of unknown function 10.780 0.055
    SOL1 Suppressor Of Los1-1, Protein with a possible role in tRNA export 10.725 0.026
    NAB6 Nucleic Acid Binding protein, Putative RNA-binding protein 10.674 0.013
    YPR148C Gene of unknown function 10.614 0.027
    ALP1 Arginine transporter 10.598 0.046
    CUT097 SUT or CUT 10.597 0.046
    ATG33 AuTophaGy related, Mitochondrial mitophagy-specific protein 10.585 0.030
    YOR316C-A Gene of unknown function 10.547 0.025
    SOG2 Key component of the RAM signaling network; required for proper cell morphogenesis and cell separation after mitosis 10.546 0.039
    MCM6 MiniChromosome Maintenance, Protein involved in DNA replication 10.531 0.019
    SUT230 SUT or CUT 10.507 0.010
    SUT419 SUT or CUT 10.398 0.027
    TIF11 Translation Initiation Factor 10.334 0.024
    TAF5 TATA binding protein-Associated Factor, involved in RNA polymerase II transcription initiation and in chromatin modification 10.328 0.027
    PHO91 PHOsphate metabolism, Low-affinity vacuolar phosphate transporter 10.303 0.024
    AIM32 Altered Inheritance rate of Mitochondria, 2Fe-2S mitochondrial protein involved in redox quality control 10.271 0.042
    ENO2 ENOlase, Enolase II, a phosphopyruvate hydratase 10.260 0.050
    UBA2 UBiquitin Activating, Subunit of heterodimeric nuclear SUMO activating enzyme E1 with Aos1p 10.215 0.030
    PUS5 PseudoUridine Synthase 10.197 0.030
    ERG1 ERGosterol biosynthesis, Squalene epoxidase 10.139 0.013
    SUT311 SUT or CUT 10.130 0.012
    KSS1 Kinase Suppressor of Sst2 mutations, Mitogen-activated protein kinase (MAPK) 10.116 0.039
    MRP10 Mitochondrial Ribosomal Protein, Mitochondrial ribosomal protein of the small subunit 10.099 0.023
    CUT598 SUT or CUT 10.099 0.046
    CUT188 SUT or CUT 10.073 0.026
    YOR238W Gene of unknown function 10.023 0.025
    EMW1 Essential for Maintenance of the cell Wall, Essential conserved protein with a role in cell wall integrity 15.549 0.071
    BNA7 Biosynthesis of NAD, Formylkynurenine formamidase 14.863 0.071
    SNR63 Small Nucleolar RNA, C/D box small nucleolar RNA (snoRNA) 14.717 0.071
    CCT3 Chaperonin Containing TCP-1, Subunit of the cytosolic chaperonin Cct ring complex 14.647 0.071
    PRY2 Pathogen Related in Yeast, Sterol binding protein involved in the export of acetylated sterols 14.548 0.071
    MAL11 MALtose fermentation, High-affinity maltose transporter (alpha-glucoside transporter) 14.484 0.071
    KRS1 Lysyl (K) tRNA Synthetase 14.290 0.072
    RAI1 Rat1p Interacting Protein, Nuclear decapping endonuclease 14.254 0.071
    SUT784 SUT or CUT 13.682 0.071
    YPR148C Gene of unknown function 13.572 0.071
    YEL1 Yeast EFA6-Like, Guanine nucleotide exchange factor specific for Arf3p 13.417 0.096
    CUT832 SUT or CUT 13.118 0.071
    NMA2 Nicotinamide Mononucleotide Adenylyltransferase 13.116 0.071
    VPS27 Vacuolar Protein Sorting, Endosomal protein that forms a complex with Hse1p 12.963 0.071
    SUT428 SUT or CUT 12.841 0.089
    PEX29 PEroXisome related, ER-resident protein involved in peroxisomal biogenesis 12.477 0.071
    YLR446W Gene of unknown function 12.369 0.071
    WBP1 Wheat germ agglutinin-Binding Protein, Beta subunit of the oligosaccharyl transferase glycoprotein complex 12.078 0.087
    AVT2 Amino acid Vacuolar Transport, Putative transporter 10.965 0.071
    CUT854 SUT or CUT 10.873 0.093
    TRM10 Transfer RNA Methyltransferase, methylates the N-1 position of guanine at position 9 in tRNAs 10.442 0.099
    Gene to be preferably combined with the particularly preferred selection
    PDI1 Protein Disulfide Isomerase 12.524 0.072
  • 4. Genes or SUTs or CUTs that were inactivated/repressed after statistical and enrichment analysis - particularly preferred selection
    Gene (common name, SUT or CUT or systematic designation) Name and function (if known) logFC FDR
    TLG2 T-snare affecting a Late Golgi compartment, Syntaxin-like t-SNARE 13.51 0.010
    CUT901 SUT or CUT 11.72 0.009
    ATG33 AuTophaGy related, Mitochondrial mitophagy-specific protein 11.53 0.009
    THR4 THReonine requiring, Threonine synthase 11.49 0.009
    YDR262W Gene of unknown function 10.92 0.009
    CMC1 Cx9C Mitochondrial protein necessary for full assembly of Cytochrome c oxidase, Copper-binding protein of the mitochondrial intermembrane space 10.86 0.009
    MRP17 Mitochondrial ribosomal protein of the small subunit 10.20 0.019
    NDC1 Nuclear Division Cycle, Subunit of the transmembrane ring of the nuclear pore complex (NPC) 14.18 0.079
    PET100 PETite colonies, Chaperone that facilitates the assembly of cytochrome c oxidase 12.69 0.086
    NIP7 Nuclear ImPort, Nucleolar protein required for 60S ribosome subunit biogenesis 12.54 0.086
    VHT1 Vitamin H Transporter, High-affinity plasma membrane H+-biotin (vitamin H) symporter 12.31 0.086
    SUT685 SUT or CUT 12.07 0.086
    BNI5 Bud Neck Involved, Linker protein responsible for recruitment of myosin to the bud neck 11.96 0.086
    SNA3 Sensitivity to NA+, Protein involved in efficient MVB sorting of proteins to the vacuole 11.93 0.086
    EGH1 Cryptococcus neoformans EGCrP2 Homolog, Steryl-beta-glucosidase with broad specificity for aglycones 11.81 0.086
    MRP4 Mitochondrial ribosomal protein of the small subunit 11.67 0.086
    POB3 POl1 Binding, Subunit of the heterodimeric FACT complex (Spt16p-Pob3p) 10.87 0.086
    PIB2 PtdIns(3)p-Binding, Phosphatidylinositol 3-phosphate binding protein 10.80 0.086
    SUT317 SUT or CUT 10.74 0.086
    NTO1 NuA Three Orf, Subunit of the NuA3 histone acetyltransferase complex 10.62 0.086
  • 5. Genes that were overexpressed - most preferred selection
    Gene (common name, SUT or CUT or systematic designation) Name and function (if known) logFC FDR
    MIC19 Component of the MICOS complex 13.883 0.036
    TOM22 Translocase of the Outer Mitochondrial membrane; responsible for initial import of mitochondrially directed proteins 13.781 0.008
    NKP1 Non-essential Kinetochore Protein 13.389 0.012
    DML1 Drosophila melanogaster Misato-Like protein, Essential protein involved in mtDNA inheritance 13.307 0.014
    CUT859 SUT or CUT 13.152 0.033
    GAL80 GALactose metabolism, Transcriptional regulator involved in the repression of GAL genes 12.170 0.008
    APM3 clathrin Adaptor Protein complex Medium chain 12.088 0.020
    COQ10 COenzyme Q, Coenzyme Q (ubiquinone) binding protein 12.048 0.025
    BLM10 BLeoMycin resistance, Proteasome activator 12.008 0.030
    MDH1 Malate DeHydrogenase, Mitochondrial malate dehydrogenase 11.915 0.008
    EMW1 Essential for Maintenance of the cell Wall, Essential conserved protein with a role in cell wall integrity 15.549 0.071
    BNA7 Biosynthesis of NAD, Formylkynurenine formamidase 14.863 0.071
    SNR63 Small Nucleolar RNA, C/D box small nucleolar RNA (snoRNA) 14.717 0.071
    CCT3 Chaperonin Containing TCP-1, Subunit of the cytosolic chaperonin Cct ring complex 14.647 0.071
    PRY2 Pathogen Related in Yeast, Sterol binding protein involved in the export of acetylated sterols 14.548 0.071
    MAL11 MALtose fermentation, High-affinity maltose transporter (alpha-glucoside transporter) 14.484 0.071
    KRS1 Lysyl (K) tRNA Synthetase 14.290 0.072
    RAI1 Rat1p Interacting Protein, Nuclear decapping endonuclease 14.254 0.071
    SUT784 SUT or CUT 13.682 0.071
    YPR148C Gene of unknown function 13.572 0.071
    YEL1 Yeast EFA6-Like, Guanine nucleotide exchange factor specific for Arf3p 13.417 0.096
    CUT832 SUT or CUT 13.118 0.071
    NMA2 Nicotinamide Mononucleotide Adenylyltransferase 13.116 0.071
    VPS27 Vacuolar Protein Sorting, Endosomal protein that forms a complex with Hse1p 12.963 0.071
    SUT428 SUT or CUT 12.841 0.089
    PEX29 PEroXisome related, ER-resident protein involved in peroxisomal biogenesis 12.477 0.071
    YLR446W Gene of unknown function 12.369 0.071
    WBP1 Wheat germ agglutinin-Binding Protein, Beta subunit of the oligosaccharyl transferase glycoprotein complex 12.078 0.087
    Gene to be preferably combined with the most preferred selection
    PDI1 Protein Disulfide Isomerase 12.524 0.072
  • 6. Genes or SUTs or CUTs that were inactivated/repressed after statistical and enrichment analysis - most preferred selection
    Gene (common name, SUT or CUT or systematic designation) Name and function (if known) logFC FDR
    TLG2 T-snare affecting a Late Golgi compartment, Syntaxin-like t-SNARE 13.51 0.010
    CUT901 SUT or CUT 11.72 0.009
    ATG33 AuTophaGy related, Mitochondrial mitophagy-specific protein 11.53 0.009
    THR4 THReonine requiring, Threonine synthase 11.49 0.009
    NDC1 Nuclear Division Cycle, Subunit of the transmembrane ring of the nuclear pore complex (NPC) 14.18 0.079
    PET100 PETite colonies, Chaperone that facilitates the assembly of cytochrome c oxidase 12.69 0.086
    NIP7 Nuclear ImPort, Nucleolar protein required for 60S ribosome subunit biogenesis 12.54 0.086
    VHT1 Vitamin H Transporter, High-affinity plasma membrane H+-biotin (vitamin H) symporter 12.31 0.086
    SUT685 SUT or CUT 12.07 0.086
  • Preferred are further genes or SUTs or CUTs that are selected from the group of genes or SUTs or CUTs having a value of logFC/FDR of more than 200, more preferred of more than 300, and most preferred of more than 500, based on the values herein.
    • References as cited
    1. 1. Martinez Ruiz, J.; Liu, L.; Petranovic, D. (2012) "Pharmaceutical protein production by yeast: towards production of human blood proteins by microbial fermentation". Current Opinion in Biotechnology, vol. 23(6), pp. 965-971.
    2. 2. Falch EA. Industrial enzymes--developments in production and application. Biotechnol Adv. 1991;9(4):643-58. doi: 10.1016/0734-9750(91)90736-f. PMID: 14542053.
    3. 3. Demain AL, Vaishnav P. Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv. 2009 May-Jun;27(3):297-306. doi: 10.1016/j.biotechadv.2009.01.008. Epub 2009 Jan 31. PMID: 19500547.
    4. 4. Zahrl RJ, Gasser B, Mattanovich D, Ferrer P. Detection and Elimination of Cellular Bottlenecks in Protein-Producing Yeasts. Methods Mol Biol. 2019;1923:75-95. doi: 10.1007/978-1-4939-9024-5_2. PMID: 30737735
    5. 5. Parapouli M, Vasileiadis A, Afendra AS, Hatziloukas E. Saccharomyces cerevisiae and its industrial applications. AIMS Microbiol. 2020 Feb 11;6(1):1-31. doi: 10.3934/microbiol.2020001. PMID: 32226912; PMCID: PMC7099199.
    6. 6. Dominguez AA, Lim WA, Qi LS. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol. 2016 Jan;17(1):5-15. doi: 10.1038/nrm.2015.2. Epub 2015 Dec 16. PMID: 26670017; PMCID: PMC4922510.

Claims (15)

  1. A yeast or filamentous fungal cell producing at least one secreted protein of interest, wherein said cell comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, VHS2, ASA1, TRP4, YPS7, CUT824, YOR318C, PRM7, ERV46, FIT2, GPM3, CUT892, SRN2, SUT643, CUT461, THR4, GMH1, SOL1, NAB6, YPR148C, ALP1, CUT097, ATG33, YOR316C-A, SOG2, MCM6, SUT230, SUT419, TIF11, TAF5, PHO91, AIM32, ENO2, UBA2, PUS5, ERG1, SUT311, KSS1, MRP10, CUT598, CUT188, YOR238W, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1 RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, WBP1, AVT2, CUT854, TRM10, SLX9, YPL077C, PET122, TFG2, PUN1, CUT152, AIR2, CUT571, RPS26B, RRT6, RPC19, URA3, YGR045C, SMC3, PNG1, THI6, MEU1, CUT239, NSE4, SUT074, AAH1, RMD5, CUT607, ACS1, MNN1, ARH1, YHR140W, CET1, RRB1, YLR342W-A, RPS22B, CHS5, YIL165C, SUT093, LPX1, NCA3, EFG1, NBP35, CUT765, MSL1, SCD6, ATG42, CHS6, COQ2, RPO31, MKK1, HED1, PBP2, BET5, CUT678, YGR021W, SUT474, YGL159W, IRC21, VHR1, SPP1, PRP43, ZRT1, YLR041W, SUT711, COX18, CBP6, SUT575, CLG1, CUT213, QCR10, SNR3, MSS2, CUT505, YOS1, SUT073, UTP21, ACA1, CUT632, RIP1, HUL5, CUT727, RPL35B, CUT184, CUT420, YFL041W-A, SUT460, ATG10, MFA1, UGX2, TRK2, CUT704, SUT083, TRE1, RVS161, LEA1, EBP2, THI80, CTI6, CUT322, XPT1, MRPL35, YPL025C, SUT737, PGA2, ULP2, MRX16, EST1, NUP100, IES3, ATG39, YMR084W, SUT428, YPL119C-A, MIN8, CUT490, SUT287, KEL3, SUT678, SEC3, SOL4, SIS2, CUT915, RRP3, ESA1, PCL8, TRX3, YKL115C, EMP65, ZDS1, CUT167, SOD1, UBR2, LSP1, SNR81, RGD3, YTP1, SMY2, CUT449, FIN1, YKL106C-A, YAR019W-A, CCH1, AYR1, SUT573, VNX1, FOL3, SUT511, GIS4, CUT743, RPL24A, HMT1, SUT333, SPP2, SUT128, SMC6, PHR1, RPS15, CUT642, GYP7, tK(CUU)K, CUT896, SLM5, CUT586, CUT158, and RRP12, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, YDR262W, CMC1, MRP17, YPT52, CUT312, MRPS5, RDR1, DAL7, RPL20A, YBR137W, RPL36B, YEL008C-A, RAX1, INP51, CUT729, UBP8, CUT258, YLR342W-A, SUT568, PEX7, MSD1, CUT136, TIM10, CUT361, snR51, TAL1, RIP1, MRP10, SUT078, MRP51, GLO3, EHD3, HER1, NMA111, PBP4, MFB1, IKI3, NDL1, SUT433, YOR238W, SUT750, QDR2, RDI1, SUT014, CUT437,
    MSC6, SUT497, YCR051W, MRPL33, RPL14A, TRM7, RNH202, RTC5, SUT027, CDC5, SUT729, YOR131C, CUT665, GLG2, SUT268, SUT705, MED4, RCR2, EFB1, RXT2, KGD1, TUP1, RNH203, YDR338C, SED1, CUT522, HIS2, SUT145, MET17, APC4, NKP2, MKK2, NDC1, PET100, NIP7, VHT1, SUT685, BNI5, SNA3, EGH1, MRP4, POB3, PIB2, SUT317, and NTO1, wherein said at least one fungal gene shows reduced expression and/or inactivation, and optionally further comprising the fungal gene PDI1, showing an increased expression and/or overexpression.
  2. The yeast or filamentous fungal cell according to claim 1, wherein said cell comprises at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1, RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, and WBP1, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, NDC1, PET100, NIP7, VHT1, and SUT685, wherein said at least one fungal gene shows reduced expression and/or inactivation, and optionally further comprising the fungal gene PDI1, showing an increased expression and/or overexpression.
  3. The yeast or filamentous fungal cell according to claim 1 or 2, wherein said genes or SUTs or CUTs are furthermore selected from the group of genes or SUTs or CUTs having a value of logFC/FDR of more than 200, more preferred of more than 300, and most preferred of more than 500, based on the values as determined herein.
  4. The yeast or filamentous fungal cell according to any one of claims 1 to 3, further comprising a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions.
  5. The yeast or filamentous fungal cell according to any one of claims 1 to 4, wherein said yeast or filamentous fungal cell is selected from the group consisting of Aspergillus spp., Trichoderma spp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces ssp., Pichia spp., Hansenula polymorpha, Fusarium spp., Neurospora spp., and Penicillium spp., preferably Saccharomyces cerevisiae.
  6. The yeast or filamentous fungal cell according to any one of claims 1 to 5, wherein said at least one secreted protein of interest also shows an increased expression and/or overexpression.
  7. The yeast or filamentous fungal cell according to any one of claims 1 to 6, wherein said at least one fungal gene showing increased expression and/or overexpression and/or showing reduced expression and/or inactivation is a native gene and/or is a recombinant gene, wherein preferably said recombinant gene is integrated into the genome as an expression cassette and/or extrachromosomally expressed, preferably using a replicative expression vector.
  8. The yeast or filamentous fungal cell according to any one of claims 1 to 7, wherein the cell furthermore comprises at least one additional recombinant secretion promoting gene, for example a gene for a chaperone, for a foldase and/or for a glycosylation-promoting protein.
  9. The yeast or filamentous fungal cell according to any one of claims 1 to 8, wherein the increased expression and/or overexpression or reduced expression and/or inactivation of the at least one fungal gene or the at least one additional recombinant secretion promoting gene is constitutive or inducible.
  10. The yeast or filamentous fungal cell according to any one of claims 1 to 9, wherein the cell produces the at least one secreted protein to about 30% or more, or about 40% or more, preferably about 50% or more, more preferably to about 75% or more, when compared to a control yeast or filamentous fungal cell.
  11. A method for producing a secreted protein in a yeast or filamentous fungal cell, comprising the steps of i) providing a yeast or filamentous fungal cell producing at least one secreted protein of interest according to any one of claims 1 to 10, ii) culturing said yeast or filamentous fungal cell in suitable culture medium, and iii) isolating said secreted protein from said culture medium, and optionally further comprising suitably inducing the increased expression and/or overexpression or reduced expression and/or inactivation of the at least one fungal gene.
  12. The method according to claim 11, wherein preferably about 30% or more, or about 40% or more, preferably about 50% or more, more preferably to about 75% or more of said at least one secreted protein is produced, when compared to the production of a control yeast or filamentous fungal cell.
  13. A method for producing a yeast or filamentous fungal cell producing at least one secreted protein of interest, comprising introducing into said cell producing at least one secreted protein of interest at least one fungal gene selected from the group consisting of MIC19, TOM22, NKP1, DML1, CUT859, GAL80, APM3, COQ10, BLM10, MDH1, EMW1, BNA7, SNR63, CCT3, PRY2, MAL11, KRS1, RAI1, SUT784, YPR148C, YEL1, CUT832, NMA2, VPS27, SUT428, PEX29, YLR446W, and WBP1, wherein said at least one fungal gene shows increased expression and/or overexpression, and/or wherein said cell comprises at least one fungal gene selected from the group consisting of TLG2, CUT901, ATG33, THR4, NDC1, PET100, NIP7, VHT1, and SUT685, wherein said at least one fungal gene shows reduced expression and/or inactivation, and optionally further introducing into said cell a fungal gene selected from the group consisting of THR4, MRP10, RIP1, YLR342W-A, ATG33, and YOR238W, either showing an increased expression and/or overexpression or reduced expression and/or inactivation, depending on the experimental conditions, and/or optionally further introducing into said cell the fungal gene PDI1, showing an increased expression and/or overexpression.
  14. The method according to any one of claims 11 to 13, wherein said at least one fungal gene is integrated into the genome as an expression cassette and/or extrachromosomally expressed, preferably using a replicative expression vector.
  15. Use of a yeast or filamentous fungal cell according to any one of claims 1 to 10 for producing at least one secreted protein of interest.
EP21213084.3A 2021-12-08 2021-12-08 Improved production of secreted proteins in fungal cells Pending EP4194560A1 (en)

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